Anthocyanin production in a callus line of Panax sikkimensis Ban

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“Rising”Intonationon“Falling”Tones

“Rising” Intonation on “Falling” TonesMasayuki GibsonCornell UniversityThe nature of the interaction between sentence level intonation and lexical tone varies from language to language. This is clearly evident in how “rising” intonation (an intonational contour that is perceived as rising) interacts with the lexical tones on words near the right edge of the utterance in different languages. Adherents of the ToBI-style notation (Pierrehumbert and Beckman 1988, e.g.) analyze such utterances as bearing a H boundary tone at the right edge. While this is a reasonable analysis, it is a necessarily language-dependent one that is insufficient to capture the typological variation that is apparent upon inspection of data from multiple languages. Yi Xu’s (2005) PENTA model treats tones and intonation as separate functions that are implemented by the Phonetics in parallel. This model assumes that tone and intonation do not interact in the Phonology. Results from production and perception experiments that were conducted in several languages for this study, including Shiga Japanese, Mandarin, Cantonese, and North Kyeongsang Korean suggest that any model of speech melody must allow for both language-specific phonetic implementation and for the interaction of tone and intonation in the Phonology.Shiga Japanese: Unlike in Tokyo Japanese, finally-accented words in SJ retain the drop in pitch associated with the accent on a final light syllable, thus maintaining the contrast between finally-accented and unaccented words. The realization of the pitch drop in this dialect does not seem to require a lengthening of that final mora/syllable. Meanwhile, when pronounced with an echo question intonation, the final mora of a finally-accented word still displays a sharp rise after the drop associated with the accent. The realization of this rise is accompanied by a drastic lengthening of the last mora (doubling its length in most cases). (See Figure 1a) Mandarin: Echo questions in Mandarin (Putonghua) are characterized by a raising of the overall pitch level that causes a final lexical falling tone to be in a higher range than in a declarative utterance (but falling nonetheless). Yuan (2004) shows that in echo questions the F0 is shifted up from the start of the utterance and that the pitch range of the final syllable is increased. Results from the present production study confirm this. Yuan’s observation that the effect of the echo question intonation on the tones is tone-specific is also corroborated; the first and second tones seem simply to be shifted upwards, whereas the third tone gets pulled down just as low as a declarative third tone but then ends much higher and the beginning of the fourth tone gets shifted upwards to a greater degree than the end. Overall, we don’t see duration effects in Mandarin that are comparable to those seen in SJ, but we do see a slight lengthening of the final syllable for one speaker that could be attributed to phonetic marking of focus (see Chen 2002). (See Figure 1b)Cantonese: In Hong Kong Cantonese, the intonational rise of an echo question is realized on the final syllable. The results of the present study show that, unlike in Shiga Japanese, a final falling tone in Cantonese does not complete its fall before the rise is initiated (c.f. Wu 1990, who reports that “the rise starts after the fall” and Yip 2002, who claims that the tone starts where it would in a declarative context and ends “high”). Like in Mandarin, we see some tone-specific effects. Also like in Mandarin, the duration of the final syllable is not affected to the degree that we see in SJ. (See Figure 1c)North Kyeongsang Korean: Despite its status as a so-called “pitch accent” language, NKK behaves quite differently from Japanese when it comes to the reconciliation of a lexical HL sequence with a rising intonation. If the HL sequence falls on the last two syllables of an echo question, we still see a lower F0 on the second syllable, though it doesn’t drop as low as it does in a declarative context. This is similar to the Mandarin case. However, if the HL sequence falls on a single syllable, there is no fall; the pitch simply rises and keeps rising. (See Figure 1d) An adequate model of speech melody must minimally include a phonological component and a phonetic component. The phonological component must be able to do “repairs” like tone deletion (to handle NKK) and TBU lengthening (to handle SJ). The “rising” intonation scheme must also be sensitive to the tonal category (to handle the tone-specific phonetic implementation in Cantonese and Mandarin), rendering parallel implementation of tones and intonation impossible to maintain. Such a model would not only give us better descriptive power but would also go further than the other models mentioned above in accounting for cross-linguistic differences in perception. For example, Cantonese speakers are better able to identify the sentence type of an echo question but not as good at identifying the final lexical tone, whereas the reverse is true for Mandarin speakers. This asymmetry is likely due to the fact that, while the tone-specific implementation of intonation reinforces differences among the tones in Mandarin, it nearly neutralizes several of the tones in Cantonese.a. SJ: + “rising” intonationb. Mandarin: + “rising” intonationc. Cantonese: + “rising” intonationd. NKK (1 syll): + “rising” intonatione. NKK (2 syll): + “rising” intonationFigure 1: A schematic representation of the interaction of lexical HL tones with “rising” intonation associated with echo questions in (a) Shiga Japanese, (b) Mandarin, (c) Cantonese, (d,e) North Kyeonsang Korean.。

Expression Analysis of Anthocyanin Biosynthetic Genes in Different

Expression Analysis of Anthocyanin Biosynthetic Genes in Different Colored Sweet Cherries (Prunus avium L.)During Fruit DevelopmentYun Liu •Xinjie Shen •Kai Zhao •Yue Ben •Xinwei Guo •Xiaoming Zhang •Tianhong LiReceived:26February 2013/Accepted:28May 2013/Published online:23July 2013ÓSpringer Science+Business Media New York 2013Abstract To identify the key enzymes involved in anthocyanin synthesis in sweet cherry (Prunus avium L.),the differences in anthocyanin biosynthetic gene expres-sions were investigated in the samples (mix of peel and ﬂesh)of a red-colored cultivar (‘Hongdeng’)and a bicol-ored cultivar (‘Caihong’)during fruit development.The expression of six anthocyanin synthetic genes in cherry (PacCHS ,PacCHI ,PacF3H ,PacDFR ,PacANS ,and PacUFGT )was analyzed by quantitative real-time PCR and Western blot analysis.Meanwhile,the changes in anthocyanin contents were measured by ultra-performance liquid chromatography.The expression of anthocyanin synthetic genes and the anthocyanin contents were much higher in ‘Hongdeng’than in ‘Caihong’fruits.Gene tran-scription and translation and anthocyanin accumulation all started approximately at the end of the pit-hardening periodand reached a maximum at maturation.All six genes were signiﬁcantly correlated with anthocyanin accumulation in ‘Hongdeng’and PacCHS had the highest direct effect.However,only PacUFGT was signiﬁcantly correlated with anthocyanin accumulation in ‘Caihong’.Anthocyanin biosynthesis in sweet cherry seems to be regulated mostly at the transcript levels.CHS appears to be the key enzyme involved in anthocyanin synthesis in ‘Hongdeng’,while UFGT is involved in anthocyanin synthesis in ‘Caihong’fruits.Keywords Sweet cherries ÁAnthocyanin synthetic genes ÁqRT-PCR ÁWestern blotting ÁUltra-performance liquid chromatographyIntroductionSweet cherry (Prunus avium L.)is a very popular tem-perate fruit.It contains high levels of anthocyanins (Kim and others 2005;Usenik and others 2008),which are beneﬁcial to humans because they have antioxidant func-tions,are able to protect against coronary heart disease,and are also involved in the body’s defense against pathogens and ultraviolet radiation (Gronbaek and others 1995;Knekt and others 1997;Guendez and others 2005;Nhukarume and others 2010).Knowledge of the molecular mechanism of anthocyanin biosynthesis is of great importance in order to improve anthocyanins in food crops.Chalcone synthase (CHS)is the ﬁrst committed enzyme in the anthocyanin pathway.It catalyzes the synthesis of tetrahydrocannabinol (THC)from one molecule of 4-cou-maroyl CoA and three molecules of malonyl CoA (Tanaka and others 2008).Fukusaki and others (2004)reported that ﬂower color in the garden plant Torenia hybrid wasY.Liu ÁX.Shen ÁK.Zhao ÁX.Guo ÁT.Li (&)Department of Pomology,Key Laboratory of Stress Physiology and Molecular Biology for Tree Fruits of Beijing,College of Agronomy and Biotechnology,China Agricultural University,Beijing 100193,People’s Republic of China e-mail:lith@ Y.Liue-mail:whbeuty@Y.BenBeijing No.101Middle School,Haidian,Beijing 100091,People’s Republic of ChinaX.ZhangInstitution of Forestry and Pomology,Beijing Academy of Agriculture and Forestry Science,Beijing 100093,People’s Republic of ChinaT.LiNo.2,Yuanmingyuan West Road,Haidian,Beijing 100194,People’s Republic of ChinaJ Plant Growth Regul (2013)32:901–907DOI 10.1007/s00344-013-9355-3successfully modulated by RNA interference(RNAi)of the CHS gene,with which the original blueﬂower color changed to white and pale colors.THC is isomerized to the colorless(2S)-naringenin by chalcone isomerase(CHI).Li and others(2006)found that transgenic tobacco plants overexpressing sense SmCHI produced up toﬁve times more totalﬂavonoids compared to wild-type tobacco plants.(2S)-Naringenin is hydroxylated at the3-position by ﬂavanone3-hydroxylase(F3H)to form dihydroﬂavonol [(2R,3R)-dihydrokaempferol,and so on].A study showed that the F3H gene was expressed in any grape plant tissues that accumulateﬂavonoids,in particular,the skin of rip-ening red berries,which synthesizes most of the anthocy-anins(Castellarin and others2006).Dihydroﬂavonols are reduced to corresponding3,4-cisleucoanthocyanidins by the action of dihydroﬂavonol4-reductase(DFR).Northern blot analysis showed that the expressions of F3H and DFR were higher inﬂowers of alfalfa(Medicago sativa L.)than in roots and nodules;the purpleﬂowers contained more anthocyanins(Charrier and others1995).Anthocyanidin synthase(ANS,also called leucoanthocyanidin dioxygen-ase)catalyzes the synthesis of colored anthocyanidins.The study conducted by Jaakola and others(2002)demon-strated the coordinated expression ofﬂavonoid biosynthetic genes(including ANS)in relation to the accumulation of anthocyanins,proanthocyanidins,andﬂavonols in the developing fruits of bilberry.Anthocyanidins are initially 3-glucosylated by the action of UDP-glucose:ﬂavonoid(or anthocyanidin)3GT and then turned into anthocyanins. The synthesis of anthocyanin in grape involves the expression of CHS,F3H,DFR,ANS,and UFGT genes. During grape ripening(after veraison),UFGT activity was concomitant with the increase in anthocyanin content (Mori and others2005).However,UFGT expression was not detected in white grapes(Boss and others1996).Although many studies have focused on the anthocyanin synthetic genes such as CHS,CHI,F3H,DFR,ANS,and UFGT in apples,pears,and so on(Espley and others2007; Mano and others2007;Feng and others2010),there are few reports about these genes in cherries.Wang and others (2010a)compared ANS and CHS expression levels in the cherry fruit of‘Stella’(a red fruit cultivar)with those in ‘Rainier’(a bicolored cultivar)during fruit development.Our previous research indicated differences in constit-uents and contents of phenolic components between red-colored cherries and bicolored cherries(Liu and others 2011).In this study,to identify the key enzymes involved in anthocyanin synthesis in sweet cherry,the anthocyanin content and the expression of anthocyanin synthetic genes at the mRNA and protein levels were investigated in the red-colored cultivar‘Hongdeng’and the bicolored cultivar ‘Caihong’during the fruit development period.Materials and MethodsPlant MaterialsThe sweet cherry(Prunus avium L.)cultivars‘Hongdeng’and‘Caihong’were cultivated at the Beijing Institute of Forestry and Pomology(Beijing,China)underﬁeld con-ditions.The fruits of the red-colored cultivar‘Hongdeng’had red-coloredﬂesh and peel at maturation,whereas the bicolored‘Caihong’had only a small amount of red color in the peel.In2010and2011,cherry fruits at different developmental stages were collected.The collecting dates were5,14,19,22,25,28,31,34,37,40,and43day after full bloom(DAFB).The seeds were immediately separated from peel andﬂesh.The peel andﬂesh of the cherry are very difﬁcult to separate from each other,especially in young fruit,so a mixture of peel andﬂesh was used as samples for all the experiments.All the samples were frozen in liquid nitrogen and stored at-80°C for sub-sequent analysis.Extraction of AnthocyaninsAnthocyanins were extracted according to the method of Liu and others(2011).The cherry sample(0.5g)was ground into a homogenate and extracted using7.5ml of methanol solution(containing0.5%hydrochloric acid)at -20°C for24h.After centrifugation(10,0009g,4°C)for 15min,the supernatant was collected for analysis.The anthocyanin contents were measured immediately after extraction to avoid degradation.The extracted supernatant wasﬁltered through a0.22-l mﬁlter and then10l l was injected into the ultra-performance liquid chromatography (UPLC)analyzer.Quantiﬁcation of the AnthocyaninsThe UPLC system used was an ACQUITY UPLC series (Waters,Milford,MA,USA)with a diode array detector (DAD).The anthocyanins were analyzed at530nm. Chromatographic separations were performed with an ACQUITY UPLC BEH C18(Waters)column (2.19100mm,i.d.=1.7l m)placed in a column oven at 35°C.The method used was that of Liu and others(2011) with some modiﬁcations.Two solvents[acetonitrile(sol-vent A)and0.1%triﬂuoroacetic acid in water(solvent B)] were used with a constantﬂow rate of0.2ml/min.For the elution program,the following proportion of solvent B was used:0–8min,95–75%B;8–12min,75–60%B;12–15 min,60–10%B;and15–17min,10–95%B.The antho-cyanin contents were assessed from peak areas and calcu-lated as equivalents of standards.Cyanidin-3O-rutinoside(Sigma-Aldrich Chemical Co.,St.Louis,MO,USA)was used as the equivalents of standards.RNA Extraction and Quantitative Real-timePCR(qRT-PCR)AnalysisTotal RNA was extracted from1.0g of peel andﬂesh using the hot borate method(Wan and Wilkins1994).Synthesis ofﬁrst-strand cDNA from1l g of total RNA isolated from each sample was conducted using M-MLV reverse trans-criptase(Promega,Madison,WI,USA)with an oligo-dT primer and a random primer.The cDNA was used as a template for qRT-PCR. Ampliﬁcation of speciﬁc regions of targeted genes and real-time detection of amplicon production were conducted using an ABI7500qRT-PCR system(Applied Biosystems, Carlsbad,CA,USA)with the primers(Table1)designed according to the sequences of the anthocyanin biosynthetic genes in sweet cherries(PacCHS,PacCHI,PacF3H, PacDFR,PacANS,PacUFGT).The following genes were cloned by our laboratory and their GenBank accession numbers are PacCHS,JF748833;PacCHI,F740091; PacF3H,JF740092;PacDFR,JF740093;PacANS, JF740094and PacUFGT,JF740090.Ultra SYBRÒMixture(with ROX)(CWBIO,Beijing, China)was used to perform the qRT-PCR.The reaction solutions contained20l M of forward primer,20l M of reverse primer,2ng of cDNA template,10l l of Ultra SYBR Mixture and6l l of water for a total volume of 20l l.The reactions were carried out under the following conditions:95°C for5min(1cycle)and95°C for10s and60°C for30s(40cycles).A melting curve analysis was also undertaken.PCR ampliﬁcation of a single product of the correct size for each gene was conﬁrmed by agarose gel electrophoresis and double-strand sequencing.Protein Extraction and Western BlottingTotal proteins were extracted according to the method of Zheng and others(2009)with modiﬁcations.The extracted proteins were separated by SDS-PAGE in a10%polyacrylamide gel as described by Laemmli(1970).The same amount of total protein(8l g)was loaded per lane. After electrophoresis,the proteins were electrotransferred to nitrocellulose(NC)(0.45mm,PALL)using a transfer apparatus(Bio-Rad,Hercules,CA,USA)according to Isla and others(1998).For Western blot analysis,immuno-logical detection of proteins on the NC membrane was carried out using primary polyclonal CHS,CHI,F3H, DFR,ANS,and UFGT antibodies in a1/200dilution with alkaline phosphatase conjugated anti-rat IgG antibody from goat(CWBIO,Beijing,China;1/1,000dilution)as a sec-ondary antibody at25°C.Then the membrane was stained with5ml of3,3,5,5-tetramethylbenzidine for5–30min. The color change was monitored during the staining period. Statistical AnalysisResults presented in the tables andﬁgures are shown with standard deviations.Correlation analysis and path coefﬁ-cient analysis were statistically assessed using SPSS ver.13.0and SigmaPlot8.0.The test of statistical signiﬁcance was based on the total error criteria with a conﬁdence level of95.0and99.0%.ResultsChanges in Anthocyanin Contents during Sweet Cherry Fruit DevelopmentAnthocyanin levels were measured in each sweet cherry fruit sample.No anthocyanins were detected in‘Hongd-eng’or‘Caihong’sweet cherry fruits before31DAFB. Soon after31DAFB,the anthocyanin contents increased rapidly,especially in the‘Hongdeng’cherry fruits(Fig.1). The highest anthocyanin content was reached about40 DAFB for‘Hongdeng’and43DAFB for‘Caihong’.The anthocyanin content in‘Hongdeng’[maximum659.0 mg/100g FW(fresh weight)]was signiﬁcantly higher than in‘Caihong’(maximum51.2mg/100g FW),which was not surprising as‘Hongdeng’is a red-colored cultivar and ‘Caihong’is a bicolored cultivar.Table1Primers used in qRT-PCR experimentsGene Forward primer Reverse primerPacCHS50-GGTGCTCGTGTTCTTGTTGTG-3050-ACTGTCGGGAAGGATGGTTTG-30PacCHI50-TCCACCGTCAGTCAAACCACC-3050-TCCAGCCCCCTCACCCCT-30PacF3H50-GATTGTGGAGGCTTGTGAGGAT-3050-GTAAATGGCTGGAGACGATGAA-30PacDFR50-CGAAGTGACCAAGCCAACAATAA-3050-GCAGAGGATGTAAACACCAGC-30PacANS50-GGGATTGGAAGAAGGGAGGC-3050-ATTTGGAACACACTTGGCAGAG-30PacUFGT50-GGTGTTTGATGTGGCTGATGG-3050-GCTGGTTGTAAAGTTGTGGGG-3018s rRNA50-AGTCGGGGGCATTCGTATTT-3050-CCCTGGTCGGCATCGTTTAT-30Changes in Gene Expression during Sweet Cherry Fruit DevelopmentTo study the molecular mechanism behind anthocyanin accumulation during fruit development,PacCHS ,PacCHI ,PacF3H ,PacDFR ,PacANS ,and PacUFGT ,which are related to sweet cherry anthocyanin synthesis,were cloned from sweet cherry fruit and antibodies to each gene were obtained.Western blot analysis and qRT-PCR were per-formed to analyze the relative-fold expression of these genes during fruit development.The results showed that the transcripts of all six genes were detected throughout the entire fruit development period,but the expression patterns were different among the genes (Fig.2).The expression of PacCHS in ‘Hongdeng’increased at 31DAFB and reacheda maximum at 40DAFB,indicating a close association with the accumulation of anthocyanin.The increase in the expression of PacCHI and PacF3H started about 3–4days later than that of PacCHS in ‘Hongdeng’and the increase in the expression of PacDFR and PacANS was even later at about 37DAFB.All the expression peaks occurred at 40DAFB.The increased expression time points for the ﬁrst ﬁve genes (PacCHS ,PacCHI ,PacF3H ,PacDFR ,and PacANS )were in the same order as the anthocyanin syn-thesis pathway.However,the sixth gene,PacUFGT ,was an exception.Its expression increased at 22DAFB,which was earlier than the start of anthocyanin accumulation.The expression patterns of these genes in ‘Caihong’were different from the patterns in ‘Hongdeng’(Fig.2).The expressions of the ﬁrst ﬁve genes (PacCHS ,PacCHI ,PacF3H ,PacDFR ,and PacANS )were almost unchanged during the entire fruit development period.Expression levels were also low compared to those in ‘Hongdeng’.Only PacUFGT had an expression maximum at the same time as the anthocyanin accumulation peak.Its expression increased at 28DAFB,which was earlier than the start of anthocyanin accumulation.The Western blot results were consistent with qRT-PCR (Fig.3).In ‘Hongdeng’cherry fruits,the CHS protein was detected at 34DAFB and CHI and F3H proteins were detected at 40DAFB.These were later than the transcript increase times.The DFR protein was detected at 37DAFB,when transcript levels had started to increase.The proteins associated with the last two genes (ANS and UFGT)were detected prior to the transcript level increases,particularly the UFGT.All six genes caused a maximum protein pro-duction peak at 40DAFB.No visible signals were obtained from ‘Caihong’except for the UFGT,which conﬁrmed the results of the qRT-PCR analysis.‘Hongdeng’ ‘Caihong’Days after full bloom152025303540451e+5PacCHS in 'Hongdeng'PacCHI in 'Hongdeng'PacF3H in 'Hongdeng'PacDFR in 'Hongdeng'PacANS in 'Hongdeng'PacCHS in 'Caihong'PacCHI in 'Caihong'PacF3H in 'Caihong'PacDFR in 'Caihong'PacANS in 'Caihong'PacUFGT in 'Caihong'PacUFGT in 'Hongdeng'Correlation Analysis and Path Coefﬁcient Analysis Correlation analysis and path coefﬁcient analysis were conducted between gene expression levels and anthocyanin content using SPSS ver.13.0.The results indicated that the expressions of all six genes were signiﬁcantly correlated with anthocyanin accumulation in ‘Hongdeng’fruits and four of them were at the 0.01level of signiﬁcance (Table 1).The results of the path coefﬁcient analysis showed that PacCHS had the highest direct effect (P 1=2.269).PacCHI showed signiﬁcantly higher positive indirect effects via PacCHS (P 21=0.9161)on anthocya-nin content in ‘Hongdeng’,whereas PacF3H (P 31=-0.5563),PacDFR (P 41=-1.2884),and PacANS (P 51=-0.4844)had negative indirect effects.Only the expres-sion level of PacUFGT (r 2=0.9371)was signiﬁcantly correlated with anthocyanin content in ‘Caihong’(Table 2).The path coefﬁcient calculation for ‘Caihong’showed that PacUFGT (P 6=1.577)had signiﬁcantly higher direct effects on anthocyanin content than the other genes.DiscussionAnthocyanin contents have been shown to differ between the red cherry cultivar and the bicolored cultivar (Usenik and others 2008;Liu and others 2011).However,the molecular basis of this variation is not clear.This study was carried out using the red-colored sweet cherry cultivar ‘Hongdeng’and the bicolored cultivar ‘Caihong’.UPLC analysis revealed that the anthocyanin content in ‘Hongd-eng’was signiﬁcantly higher than in ‘Caihong’(Fig.1).This was because the ﬂesh (mesocarp)of ‘Hongdeng’was as red as the peel (exocarp),whereas there was very little red in the peel of the ‘Caihong’fruit and none in the ﬂesh.Consistent with anthocyanin content,‘Hongdeng’was associated with higher transcriptions of the genes studied than ‘Caihong’at each fruit development stage.Takos and others (2006)reported that the transcript levels of the MdCHS ,MdF3H ,MdDFR ,MdLDOX ,and MdUFGT genes,which are required for apple anthocyanin synthesis,were lower in the peels of nonred than in red-peeled cul-tivars.A similar study in cherries was also carried out by Wang and others (2010a ).The results showed that ANS and CHS expression levels were much higher in the cherry fruits of ‘Stella’(a red fruit cultivar)compared to ‘Rainier’(a bicolored cultivar)during the last two stages of fruit development.Their results were conﬁrmed by this study.The expression levels of all six genes studied were much higher in ‘Hongdeng’than in ‘Caihong’(Fig.2).Our results also showed that the ﬁrst ﬁve genes (PacCHS ,PacCHI ,PacF3H ,PacDFR ,and PacANS )were barely expressed during the entire fruit development period in ‘Caihong’(Fig.2).A similar ﬁnding by Kim and others (2003)showed that the transcription of genes during the later stages of anthocyanin biosynthesis in green apples was barely detectable by RNA gel blot analysis.The two types of cherry fruits used in this study both started accumulating anthocyanin at approximately the end of the pit-hardening period,when the fruit barely enlarges514192225283134374043DAFBAB C D E F GFig.3Western blot of the six genes in cherries.a –f The protein accumulation of CHS,CHI,F3H,DFR,ANS,and UFGT in ‘Hongdeng’cherry fruits during fruit development.g The protein accumulation of UFGT in ‘Caihong’cherry fruits during fruit development.DAFB days after full bloomTable 2Direct and indirect effects of the six major genes on anthocyanin accumulation in ‘Hongdeng’fruits GenesCorrelationcoefﬁcients with anthocyanin content Direct effect trait on anthocyanin content Indirect effect traits by the genes PacCHS (1)PacCHI (2)PacF3H (3)PacDFR (4)PacANS (5)PacUFGT (6)PacCHS (1)0.8820** 2.26900.9161-0.5563-1.2884-0.48440.0260PacCHI (2)0.9976**0.9618 2.0712-0.5047-1.0949-0.45900.0232PacF3H (3)0.9840**-0.5234 2.40030.9099-1.3301-0.49890.0262PacDFR (4)0.6918*-1.3929 2.23550.8317-0.5245-0.48370.0257PacANS (5)0.9207**-0.4892 2.24690.9164-0.5683-1.20990.0248PacUFGT (6)0.7453*0.03012.05380.7391-0.4857-1.1751-0.4169*indicates signiﬁcant differences at the 0.05level of signiﬁcance (R 2=0.6319);**indicates signiﬁcant differences at the 0.01level of signiﬁcance (R 2=0.7646)and the stony endocarp(pit)undergoes hardening(DeJong and Grossman1995).Anthocyanin accumulation in both cultivars reached a peak at maturation.The anthocyanin peak for‘Caihong’was a little later than that of‘Hongd-eng’because‘Caihong’sweet cherries are harvested later than‘Hongdeng’.The increase in the transcripts of theﬁrst ﬁve genes in‘Hondeng’was closely associated with the increase in accumulation of anthocyanin,which occurred right after the pit-hardening period ended and reached a peak at fruit maturation.The protein accumulation started to increase later than the transcript accumulation.Although these results showed that the dates when proteins of the last two genes(ANS and UFGT)could be detected were about 3–7days earlier than their transcript times,the sharply increasing time points for protein production were still consistent with the increase in transcript levels.When the transcripts of the last two genes were not increasing shar-ply,they were able to produce enough proteins to be detected.Therefore,the protein amounts were determined by the transcript levels.There have been few reports about the protein accu-mulation of anthocyanin biosynthetic genes.Wang and others(2010b)detected changes in ANS protein levels during grape berry development.Although the ANS pro-tein accumulation pattern of grape was different from that of cherry in this study,both studies indicated that protein accumulation is in accord with their transcript accumula-tion.These results suggest that anthocyanin biosynthesis in sweet cherry seems to be mostly regulated at the transcript level.Although PacUFGT had a much higher expression level than the otherﬁve genes(Fig.2)and had a signiﬁcant correlation with anthocyanin contents in‘Hongdeng’(Tables2,3),transcript and protein accumulation of this gene started much earlier than the anthocyanin accumula-tion.This suggested that UFGT may promote other sub-strates that accumulated earlier than did the anthocyanins. For example,theﬂavonols were synthesized before the anthocyanins(Winkel-Shirley2001),and they also com-bined sugars,a process that is catalyzed by the UFGTs.Correlation analysis indicated that the expression of all six genes had a signiﬁcant correlation with anthocyanin accumulation in sweet cherry‘Hongdeng’.A previous report conﬁrmed that the expression of cherry CHS(an early step in the anthocyanin biosynthesis pathway)and cherry ANS(a later step)was upregulated and correlated with cherry color(Wang and others2010a).However, sometimes correlation coefﬁcients give misleading results because the correlation between two variables may be due to a third factor.Therefore,it is necessary to analyze the cause-and-effect relationship between dependent and independent variables(Sidramappa and others2008).In this study,path coefﬁcient analysis(Dewey and Lu1959) was used to investigate the nature of the relationship between dependent and associated independent variables. The variables with high positive correlations and high direct effects could be used as selection criteria in selection programs(Sadeghi and others2011).In this study,PacCHS had the highest direct effect on anthocyanin accumulation in‘Hongdeng’,indicating that to obtain an increase of anthocyanin content,PacCHS should be consideredﬁrst. Our results also showed that PacCHI had positive indirect effects via PacCHS on anthocyanin content in‘Hongdeng’cherry,whereas PacF3H,PacDFR,and PacANS might inhibit these effects.For‘Caihong’cherry,PacUFGT was the only gene that had a signiﬁcant correlation with anthocyanin accumulation.Others have reported that the MYBs,which is a large family of transcription factors associated with anthocyanin pathway,play a critical reg-ulatory role in anthocyanin biosynthesis(Espley and others 2007;Feng and others2010;Wang and others2010a),and our laboratory is conducting a study to determine the role of MYBs in cherries.ConclusionThe transcription and translation levels of anthocyanin synthetic genes and anthocyanin accumulation were all much higher in the red fruit sweet cherry‘Hongdeng’thanTable3Direct and indirect effects of the six major genes on anthocyanin accumulation in‘Caihong’fruitsGenes Correlationcoefﬁcients withanthocyanin content Direct effecttrait onanthocyanin contentIndirect effect traits by the genesPacCHS(1)PacCHI(2)PacF3H(3)PacDFR(4)PacANS(5)PacUFGT(6)PacCHS(1)-0.2543-0.0595-0.5955 1.23620.0237-0.3658-0.4934 PacCHI(2)0.1917-1.4748-0.0228 3.28280.0482-1.3144-0.3273 PacF3H(3)-0.0586 3.2665-0.0215-1.51840.0589-1.4211-0.4230 PacDFR(4)0.5088-0.20680.00650.3251-0.85920.1359 1.1073 PacANS(5)0.2523-1.4297-0.0151-1.3176 3.07690.0196-0.0818 PacUFGT(6)0.9371** 1.57710.01950.3128-0.8987-0.14950.0759**indicates signiﬁcant differences at the0.01level of signiﬁcance(R2=0.7646)in the bicolored cultivar‘Caihong’.Expression mainly started approximately at the end of the pit-hardening per-iod.Anthocyanin biosynthesis in sweet cherry seems to be mostly regulated at the transcript levels.The starting time points of gene transcription and gene expression levels were different among the six anthocyanin synthetic genes. CHS could be the key enzyme for anthocyanin contents in ‘Hongdeng’,whereas UFGT plays a major role in ‘Caihong’.Acknowledgments The authors thank Professor Weidong Huang (College of Food Science and Nutritional Engineering,China Agri-cultural University)for providing the CHI and ANS antibodies.This research was supported by National Natural Science Foundation Projects(No.31171938)and National Department of Public Beneﬁt Research Foundation(No.201003021).ReferencesBoss PK,Davies C,Robinson SP(1996)Expression of anthocyanin biosynthesis pathway genes in red and white grapes.Plant Mol Biol32:565–569Castellarin SD,Gaspero GD,Marconi R,Nonis A,Peterlunger E, Paillard S,Adam-Blondon A,Testolin R(2006)Colour variation in red grapevines(Vitis vinifera L.):genomic organisation, expression ofﬂavonoid30-hydroxylase,ﬂavonoid30,50-hydrox-ylase genes and related metabolite proﬁling of red cyanidin-/blue delphinidin-based anthocyanins in berry skin.BMC Genomics 7:12–29Charrier B,Coronado C,Kondorosi 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1-s2.0-S0031942203004382-main

ReviewAnalysis and biological activities of anthocyaninsJin-Ming Kong a ,Lian-Sai Chia a,*,Ngoh-Khang Goh a ,Tet-Fatt Chia a ,R.Brouillard baNational Institute of Education,Nanyang Technological University,1Nanyang Walk,Singapore 637616,SingaporebInstitut de Chimie,Universite Louis Pasteur,1rue Blaise Pascal,Strasbourg 67008,FranceAbstractAnthocyanins are naturally occurring compounds that impart color to fruits,vegetables,and plants.They are probably the most important group of visible plant pigments besides chlorophyll.Apart from imparting color to plants,anthocyanins also have an array of health-promoting beneﬁts,as they can protect against a variety of oxidants through a various number of mechanisms.However,anthocyanins have received less attention than other ﬂavonoids,despite this.This article reviews their biological func-tions and pre-clinical studies,as well as the most recent analytical techniques concerning anthocyanin isolation and identiﬁcation.#2003Elsevier Ltd.All rights reserved.Keywords:Anthocyanin;Biological activity1.IntroductionAnthocyanins (in Greek anthos means ﬂower,and kyanos means blue)are the more important plant pigments visible to the human eye.They belong to thewidespread class of phenolic compounds collectively named ﬂavonoids.They are glycosides of polyhydroxy and polymethoxy derivatives of 2-phenylbenzopyrylium or ﬂavylium salts (Fig.1).The diﬀerences between individual anthocyanins relate to the number of hydroxyl groups,the nature and number of sugars attached to the molecule,the position of this attachment,and the nature and number of aliphatic or0031-9422/$-see front matter #2003Elsevier Ltd.All rights reserved.doi:10.1016/S0031-9422(03)00438-2Phytochemistry 64(2003)923–933/locate/phytochemContents1.Introduction...............................................................................................................................................................................9232.Recent advances in anthocyanin analysis and identiﬁcation......................................................................................................9253.Functions of anthocyanins.........................................................................................................................................................9264.Biological activities of anthocyanins..........................................................................................................................................9265.Antioxidant properties and free radical scavenging properties..................................................................................................9286.The interaction with DNA.........................................................................................................................................................9307.Conclusions................................................................................................................................................................................931Acknowledgements..........................................................................................................................................................................931References (931)*Corresponding author.Tel.:+65-67903885;fax:+65-68969414.E-mail address:lschia@.sg (L.-S.Chia).aromatic acids attached to sugars in the molecule.To date,there are 17known naturally occurring anthocy-anidins or aglycones which are listed in Tables 1.The role of anthocyanidins in plants is summarized in Tables 2.Only six anthocyanidins are common in higher plants—pelargonidin (Pg),peonidin (Pn),cyanidin (Cy),malvidin (Mv),petunidin (Pt)and delphinidin (Dp).The glycosides of the three non-methylated anthocyani-dins (Cy,Dp and Pg)are the most widespread in nature,being present in 80%of pigmented leaves,69%of fruits and 50%of ﬂowers.The distribution of the sixmostFig.1.The ﬂavylium cation.R1and R2are H,OH,or OCH 3;R3is a glycosyl or H;and R4is OH or a glycosyl.Table 1Naturally occurring anthocyanidinsSubstitution patternNameAbbreviation 3567304050Color Apigeninidin Ap H OH H OH H OH H Orange Aurantinidin Au OH OH OH OH H OH H Orange Capensinidin Cp OH OMe H OH OMe OH OMe Bluish-red Cyanidin Cy OH OH H OH OH OH H Orange-red Delphinidin Dp OH OH H OH OH OH OH Bluish-red Europinidin Eu OH OMe H OH OMe OH OH Bluish-red HirsutidinHsOH OH H OMe OMe OH OMe Bluish-red 6-Hydroxycyanidin 6OHCy OH OH OH OH OH OH H Red Luteolinidin Lt H OH H OH OH OH H Orange MalvidinMv OH OH H OH OMe OH OMe Bluish-red 5-Methylcyanidin 5-MCy OH OMe H OH OH OH H Orange-red Pelargonidin Pg OH OH H OH H OH H Orange Peonidin Pn OH OH H OH OMe OH H Orange-red Petunidin Pt OH OH H OH OMe OH OH Bluish-red Pulchellidin Pl OH OMe H OH OH OH OH Bluish-red Rosinidin Rs OH OH H OMe OMe OH H Red TricetinidinTrHOHHOHOHOHOHRedTable 2The role of anthocyanins and 3-deoxyanthocyanidins in plants PlantCompound Origin FunctionAngiosperms Senecio cruentus Cinerarin PetalsPollinationSorghum Apigeninidin Leaf sheath Phytoalexin anti-microbial antioxidants Gymnosperms Abies concolor Petunidin-3-glucoside Cone?Cyanidin-3-glucoside Pinus contorta Anthocyanin Cold toleranceLeavesPinus banksiana ?Photoinhibibition toleranceSeedlingsFernsDavallia divaricata Pelargonidin-3-p -coumaryl-glc-5-glc(monardein)Young leaves ?Ferns species Apigenidin Leaves ?MossesBryum,Splachunm Luteolinidin-5-glc Leaves ?LiverwortCephaloziella exilifoliaAnthocyanin-likeThallus?Excerpted from Cooper-Driver et al.(1998).924J.-M.Kong /Phytochemistry 64(2003)923–933common anthocyanidins in the edible parts of plants is cyanidin(50%),pelargonidin(12%),peonidin(12%), delphinidin(12%),petunidin(7%),and malvidin(7%). The following four classes of anthocyanidin glycosides are common:3-monosides,3-biosides,3,5-diglycosides and3,7-diglycosides.3-glycosides occur about two and half times more frequently than3,5-diglycosides.So,the most widespread anthocyanin is cyanidin3-glucoside. Based on several reviews to date,it is estimated that more than400anthocyanins have been found in nature. In the book entitled‘‘Anthocyanins in Fruits,Vegetables, and Grains’’(Mazza and Miniah,1993),258anthocya-nins are listed;and according to the reviews of Har-borne and Williams(1998,2001),from January1995to December1997,85new anthocyanins were recorded, whilst from January1998to December2000,some50 new anthocyanin pigments were found in plants.2.Recent advances in anthocyanin analysis andidentiﬁcationAnthocyanins are soluble in polar solvents,and they are normally extracted from plant materials by using methanol that contains small amounts of hydrochloric acid or formic acid.The acid lowers the solution’s pH value and prevents the degradation of the non-acylated anthocyanin pigments.However,as hydrochloric acid or formic acid is concentrated during the evaporation of the methanol-hydrochloric acid or methanol-formic acid solvent,pigment degradation occurs(e.g.in the extract of Azalea cv.Alice Erauw,the cyanidin-3-monosides are converted into unstable aglycone).Small amounts of acid may also cause partial or total hydro-lysis of the acyl moieties of acylated anthocyanins that are present in some plants.One report compared var-ious techniques for the extraction of anthocyanins from red grapes and demonstrated that solvents containing up to0.12mol/l hydrochloric acid can cause partial hydrolysis of acylated anthocyanins(Revilla et al., 1998).Acetone has also been used to extract anthocya-nins from several plant sources(Giusti et al.,1998, Garcia-Viguera et al.,1998).In comparison to acidiﬁed methanol,this technique allows an eﬃcient and more reproducible extraction,avoids problems with pectins, and permits a much lower temperature for sample con-centration(Garcia-Viguera et al.,1998).Solid-phase extraction(SPE)on C18(SPE)cartridges or Sephadex is commonly used for the initial puriﬁcation of the crude anthocyanin extracts.The anthocyanins are bound strongly to these adsorbents through their unsubstituted hydroxyl groups and are separated from unrelated compounds by using a series of solvents of increasing polarity.The characterization of a mixture of anthocyanins usually involves the separation and collection of each compound,and subsequent analysis by nuclear mag-netic resonance(NMR)and fast atom bombardment mass spectroscopy(FAB-MS).For the separation and structural analysis,the use of liquid chromatography–mass spectrometry(LC–MS)technique,which combines the separation of LC with the selectivity and sensitivity of the MS detector,permits the identiﬁcation of indivi-dual compounds in a mixture of compounds.Gla ssgen et al.(1992)described the use of liquid chromatography electrospray ionization mass spectrometry(LC-ESI-MS)for the identiﬁcation of anthocyanins in the plant tissue and cell cultures of Indian black carrot(Daucus carota L.ssp.sativus).Several cyanidin derivatives were identiﬁed,including acylated and non-acylated com-pounds.Recently liquid chromatography-electron impact ionization mass spectrometry(LC-EI-MS)was also used to identify the anthocyanins of Catharanthus roseus extracts(Piovan et al.,1998).Baldi et al.(1995) used LC–MS with an atmospheric pressure-ionization ion-spray interface to analyze the anthocyanins con-tained in the grape skins(Vitis vinifera L.).Nineteen derivatives of cyanidin,delphinidin,petunidin,malvidin and peonidin were identiﬁed by this ionization tech-nique.The individual mass spectra showed peaks for the molecular ions,together with a fragment corresponding to aglycone;when acylation was present,an additional fragment was detected at mass/charge values corre-sponding to the loss of acyl moiety from the molecular ion.Since Saito et al.(1983)employed FAB-MS in the structural analysis of acylated anthocyanins violanin and platyconin,many new acylated anthocyanins have been found with the help of this technique. Atmospheric-pressure ionization(API)techniques have several advantages over other MS detection meth-ods.In API-MS the ion source is located outside the MS,the ions are formed at atmospheric pressure,and then sampled into the mass spectrometer.These are soft ionization techniques(only the molecular ion is formed),although the application of a potential at the entrance of the mass spectrometer(fragment voltage) creates suitable conditions for CID,and the production of fragment ions.Two API interfaces are available commercially,namely,the atmospheric pressure chemi-cal ionization interface(APCI)and the ESI interface. Revilla et al.(1999)analyzed the anthocyanins present in extracts of grape skins and red wine with a LC–MS system equipped with an ESI interface.In the same year,da Costa et al.(1998)used a LC–MS system that was equipped with an APCI interface for the analysis of anthocyanins(3-glucosides and3-rutinosides of cyani-din and delphinidin)from blackcurrant fruit(Ribes nigrum).The molecular ion[M+],and the mass frag-ments corresponding to successive loss of the sugar residues,[M+-146]and[M+-146-162]were detected under appropriate conditions.Wang and Sporns(1999), Wang et al.(2000)used respectively matrix-assistedJ.-M.Kong/Phytochemistry64(2003)923–933925laser desorption/ionization mass spectrometry (MALDI–MS)to perform both qualitative and quanti-tative analyses of anthocyanins in wine and fruit juice, and matrix-assisted laser desorption/ionization time-of-ﬂight mass spectrometry(MALDI–TOF–MS)to ana-lyze the content of anthocyanins in various foods. Another technique recently used for anthocyanin analysis is capillary electrophoresis(CE)which has excellent mass sensitivity,high resolution,low sample consumption and minimal generation of solvent waste. Bridle et al.(1997)reported the separation of a mixture of standards,as well as strawberry and elderberry anthocyanins,by capillary zone electrophoresis(CZE). da Costa et al.(1998)published a CZE method to separate blackcurrant anthocyanins in a fused-silica capillary using acidic phosphate buﬀers[pH1.50-aceto-nitrile(2/1v/v)].The interaction of the anthocyanins with the capillary wall was evaluated by comparing the separation of anthocyanins on a fused-silica capillary column to that on a linear polyacrylamide(PLA)coated one.The results showed that there was almost no inter-action at pH1.8between the silanols on the uncoated capillary and the anthocyanins.The four anthocyanins present in the blackcurrant juice,namely cyanidin3-glucosides and3-rutinosides,delphinidin3-glucosides and3-rutinosides,were separated within the time range expected for an LC analysis,while consuming much less sample and solvent.Watanabe et al.(1998)analyzed elderberry pigments(Sambucus nigra L.)in commercial food samples(candy,juice,and jelly)using micellar electrokinetic chromatography(MEKC).The anthocya-nins,all cyanidin derivatives,were separated in less than 10min.Other important progress in anthocyanin analy-sis has been reported(Bicard et al.,1999).It concerns the analysis of anthocyanins by CZE in acidic media. Together with the above techniques,NMR,in both one and two dimensions,is a very useful technique for structural elucidation of anthocyanins.Some examples for its applications are given here.Giusti et al.(1998) reported the separation of anthocyanins from red radish (Raphanus sativus)and their structural elucidation by one-and two-dimensional NMR.Four anthocyanins were obtained:pelargonidin3-O-[2-O-(b-glucopyr-anosyl)-6-O-(trans-p-coumaroyl)-b-glucopyranoside]5-O-(6-O-malonyl-b-glucopyranoside);pelargonidin3-O-[2-O-(b-glucopyranosyl)-6-O-(trans-feruloyl)-b-gluco-pyranoside]5-O-(6-O-malonyl-b-glucopyranoside); pelargonidin3-O-[2-O-(b-glucopyranosyl)-6-O-(trans-p-coumaroyl)-b-d-glucopyranoside]5-O-(b-glucopyrano-side)and pelargonidin3-O-[2-O-(b-glucopyranosyl)-6-O-(trans-feruloyl)-b-gluco-pyranoside]5-O-(b-glucopyr-anoside).They also investigated the three-dimensional conformation of the molecule by using NOESY techni-ques,which showed proximity between the hydrogen from the cinnamic acid acylating group and the C-4of the pelargonidin.By using NMR and other techniques,Cabrita et al.(2000)identiﬁed the anthocyanin tri-saccharides of Vaccinium padifolium.Similarly,Ter-ahara et al.(2001)isolated and identiﬁed delphinidin, cyanidin3-O-b-d-galactosides and delphinidin3-O-b-d-(6-(E)-p-coumaryl)galactopyranoside from Camellia sinensis.Tanaka et al.(2001)isolated a malonylated anthocyanin from the blueﬂowers of Meconopsis,and then identiﬁed it as cyanidin3-O-[(6-O-malonyl-2-O-b-d-xy1opyranosyl)-b-d-glucopyranoside]-7-O-b-d-gluco-pyranoside using NMR spectroscopy.Toki et al.(2001) isolated three acylated anthocyanins from the scarlet ﬂowers of Anemone coronaria,which all belong to pelar-gonidin-type anthocyanins.Theﬁrst pigment was pelar-gonidin3-O-[2-(b-d-xylopyranosyl)-6-O-(malonyl)-b-d-galactopyranoside],the second pigment was pelargonidin 3-O-[2-O-(b-d-xylopyranosyl)-6-O-(methyl-malonyl)-b-d-galactopyrano-side]and the third one was(600-O-(pelar-gonidin3-O-[200-O-(b-d-xylopyranosyl)-b-d-galactopyr-anosyl][(4-O-(b-d-glucopyranosyl)-trans-caﬀeoyl)-O-tartaryl]malonate.3.Functions of anthocyaninsThe most signiﬁcant function of anthocyanins is their ability to impart color to the plants or plant products in which they occur.They play a deﬁnite role in the attraction of animals for pollination and seed dispersal, and hence they are of considerable value in the co-evo-lution of these plant-animal interactions.Anthocyanins and3-deoxyanthocyanidins however have roles inﬂow-ering plants other than as attractants.They can act as antioxidants,phytoalexins or as antibacterial agents. Anthocyanins may be important factors along with otherﬂavonoids in the resistance of plants to insect attack(Harborne,1988).For example,cyanidin3-glu-coside was shown to protect cotton leaves against the tabacco budworm(Hedin et al.,1983).In addition to their functions in plants,anthocyanins have many other uses.For example,their important function in cognitive decline and neural dysfunction has been investigated.Joseph et al.(1999)found that fruit extracts including anthocyanins were eﬀective in rever-sing age-related deﬁcits in several neural and behavioral parameters,e.g.oxotremorine enhancement of a K1-evoked release of dopamine from striatal slices,carba-chol-stimulated GTPase activity,striatal Ca buﬀering in striatal synaptosomes,motor behavioral performance on the rod walking and accelerod tasks,and Morris water maze performance.4.Biological activities of anthocyaninsAnthocyanins also possess known pharmacological properties and are used by humans for therapeutic926J.-M.Kong/Phytochemistry64(2003)923–933purposes.Following the recognition that pigment extracts are more eﬀective than O-(b-hydroxyethyl) rutin in decreasing capillary permeability and fragility and in their anti-inﬂammatory and anti-oedema activ-ities(Wagner,1985),it is possible that anthocyanins may replace rutin and its derivatives in the treatment of illnesses involving tissue inﬂammation or capillary fra-gility.The crude anthocyanin extracts of Vaccinium myrtillus have been given orally and by intravenal or intramuscular injection to reduce capillary permeability and fragility.In a study on testing the eﬀect of anthocyanins on tumors,some anthocyanins were not eﬀective in sup-pressing tumor growth(Ghiselli et al.,1998).However, an antioxidant activity study of anthocyanin fractions from Italian red wine showed that the anthocyanin fraction was the most eﬀective both in scavenging reac-tive oxygen species and in inhibiting lipoprotein oxida-tion and platelet aggregation(Ghiselli et al.,1998).This result suggests that anthocyanins could be the key component in red wine that protects against cardiovas-cular disease.Another report on the anti-tumor activity of anthocyanins was published by Kamei et al.(1998). They found that the anthocyanin fraction from red wine suppressed the growth of HCT-15cells,which are derived from human colon cancer or AGS cells from human gastric cancer.The suppression rate by the anthocyanin fraction was signiﬁcantly higher than that of the other fractions.Obi et al.(1998)examined the ability of anthocyanin obtained from the petals of H.rosasinensis to prevent carbon tetrachloride-induced acute liver damage in rats. The results showed that those rats treated with antho-cyanin and carbon tetrachloride had signiﬁcantly less hepatotoxicity(P<0.05)than those given carbon tetra-chloride alone.This was assessed by measuring the levels of serum aspartate and alanine aminotransferase activities18hours after carbon tetrachloride was given. This result suggested that H.rosasinensis anthocyanin may be protective against carbon tetrachloride-induced liver injury.Yoshimoto et al.(1999)studied the antimutagenicity of water extracts prepared from the storage roots of four varieties of sweet potato with diﬀerentﬂesh colors, using Salmonella typhimurium TA98.They found that two anthocyanin pigments puriﬁed from the purple-colored sweet potato,3-(6,60-caﬀeylferulylsophoroside)-5-glucoside of cyanidin(YGM-3)and peonidin(YGM-6),eﬀectively inhibited the reverse mutation induced by heterocyclic amines-mutagen,Trp-P-1,Trp-P-2,and IQ in the presence of rat liver microsomal activation systems. Jankowski et al.(2000a,b)reported that the adminis-tration of anthocyanin dyes from Aronia melanocarpa to rats before the intraperitoneal injections of Platelet-Activating Factor(PAF)and ceruleine had a beneﬁcial eﬀect on the development of acute experimental pancreatitis in rats.It was revealed that this was due to the reduction of pancreatic swelling and a decrease in lipid peroxidation and adenosine deaminase activity. They also examined the eﬀect of anthocyanins from Cabernet red wine on the course and intensity of symp-toms of experimental diabetes in rats(Jankowski et al. 2000a,b).The results showed that a simultaneous daily administration of anthocyanins obtained from Cabernet red wine and streptozotocin substantially decreased sugar concentrations in the urine and blood serum. These anthocyanins also inhibited the loss of body mass caused by the injection of streptozotocin.Simulta-neously,the anthocyanin pigment prevented the gen-eration of free oxygen radicals,and decreased the peroxidation of lipids.Pawlowicz et al.(2000)attempted to determine the inﬂuence of anthocyanins from chokeberries on the generation of autoantibodies to oxidize low density lipoproteins(oLAB)in pregnancies complicated by intrauterine growth retardation(IUGR).An experiment was conducted with a study group of105pregnant women(on the turn of trimester two according to LMP) with IUGR(sonographic examination results below the 5th percentile for real gestational age)who were ran-domly divided into2groups.Fifty women were admi-nistered anthocyanins and55women were given a placebo.There was a control group of60healthy preg-nant women.They then examined the level of oxidative stress measured by the serum concentration of auto-antibodies required to oxidize low density lipoproteins (oLAB).In the anthocyanin group,the oLAB titres decreased from1104Æ41mU/ml before treatment to 752Æ36mU/ml in theﬁrst month and726Æ35mU/ml in the second month,at P<0.01.In the placebo group, the oLAB titres showed a slightly increasing trend: 1089Æ37mU/ml before treatment,1092Æ42mU/ml in theﬁrst month and1115Æ43mU/ml in the second month,at P>0.05.The oLAB titres in the control group were601Æ49mU/ml before treatment,606Æ45 mU/ml in theﬁrst month,and614Æ43mU/ml in the second month,at P>0.05.The results indicated that natural antioxidants(anthocyanins)can be useful in con-trolling oxidative stress during pregnancies complicated by IUGR.Hibiscus anthocyanins(HAs),a group of natural pig-ments occurring in the driedﬂowers of Hibiscus sabdar-iﬀa L.,are used in soft drinks and herbal medicines. Their antioxidant bioactivity has been studied and it appears that HAs can signiﬁcantly decrease the leakage of lactate dehydrogenase and the formation of mal-ondialdehyde induced by a treatment of tert-butyl hydroperoxide(t-BHP).The in vivo investigation showed that the oral pretreatment of HAs before a sin-gle dose of t-BHP signiﬁcantly lowered the serum levels of hepatic enzyme markers(alanine and aspartate ami-notransferase)and reduced oxidative liver damage.TheJ.-M.Kong/Phytochemistry64(2003)923–933927histopathological evaluation of the liver revealed that hibiscus pigments reduce the incidence of liver lesions including inﬂammation,leucocyte inﬁltration,and necrosis induced by t-BHP in rats(Wang et al.,2000). In2001,Meiers et al.found that the aglycones of the most abundant anthocyanins in food,cyanidin(Cy)and delphinidin(Del),possess the ability to inhibit the growth of human tumor cells in vitro in the micromolar range.However,malvidin(Mv),an anthocyanidin typi-cally found in grapes,was less active.The aglycones preferentially inhibited the growth of the human vulva carcinoma cell line A431,overexpressing the epidermal growth-factor receptor(EGFR).The glycosides cyani-din-3-beta-d-galactoside(Cy-3-gal,idaein)and mal-vidin-3-beta-d-glucoside(Mv-3-glc,oenin)did not aﬀect tumor cell growth up to100m M.The tyrosine kinase activity of the EGFR,isolated from A431cells,was potently inhibited by Cy and Del.However,Mv and the glycosides Cy-3-gal and Mv-3-glc were inactive up to 100m ing intact cells,they also investigated the inﬂuence of anthocyanin treatment on downstream sig-naling cascades by measuring the phosphorylation of the transcription factor Elk-1.They observed that A431 cells were transiently transfected with a luciferase reporter gene construct whose expression is controlled by a MAP kinase pathway dependent phosphorylation of a GAL4-Elk-1fusion protein.Cy and Del inhibited the activation of the GAL4-Elk-1fusion protein in the concentration range where growth inhibition was observed.Thus,they concluded that the anthocyanidins Cy and Del are potent inhibitors of the EGFR,shutting oﬀdownstream signaling cascades.Nitric oxide(NO)is a diatomic free radical produced from l-arginine by constitutive and inducible nitric oxide synthase(cNOS and iNOS)in numerous mam-malian cells and tissues.It is believed that some chronic inﬂammatory diseases are associated with NO.Wang and Mazza(2002)for theﬁrst time reported that anthocyanins had strong inhibitory eﬀects on NO pro-duction after detailed study.There are also reports of anthocyanins possessing antiulcer activity(Cristoni and Magistretti,1987)and providing protection from UV radiation(Sharma,2001).5.Antioxidant properties and free radical scavenging propertiesIn1994,Tsuda et al.reported the antioxidant activity of the anthocyanin pigments cyanidin3-O-b-glucoside (C3G)and cyanidin(Cy),which was examined by using linoleic acid autoxidation,liposome,rabbit erythrocyte membranes,and rat liver microsomal systems.C3G and Cy had antioxidative activity in all systems.Cy had a stronger activity than C3G and the same activity as a-tocopherol in the liposome and rabbit erythrocyte membrane systems.In the rat liver microsomal system, Cy and C3G exhibited stronger activity than a-toco-pherol.These data suggested that the pigments may play an important role in the prevention of lipid per-oxidation of cell membranes induced by active oxygen radicals in living systems as they become dietary anti-oxidants after ingestion.In1996,Tsuda et al.investigated the antioxidative, radical scavenging,and inhibitory eﬀects on lipid per-oxidation of UV light irradiation of three anthocyanin pigments:pelargonidin3-O-beta-d-glucoside(P3G), cyanidin3-O-beta-d-glucoside(C3G),and delphinidin 3-O-beta-d-glucoside(D3G),isolated from the Phaseo-lus vulgaris L.seed coat,and their aglycones,pelargo-nidin chloride(Pel),cyanidin chloride(Cy),and delphinidin chloride(Del).They found that all the pig-ments had strong antioxidative activity in a liposomal system and reduced the formation of malondialdehyde from UVB(320–290nm)irradiation.On the other hand,the extent of antioxidative activity in a rat liver microsomal system and the scavenging eﬀect of hydroxyl radicals(.OH)and superoxide anion radicals (.O2À)were inﬂuenced by their own structures.In the same year,Koide et al.(1996)found that hydrolyzed anthocyanidins contained in grape rinds and red rice gave an elevation of S phase,which suggested a block in the step from S-phase to G2-phase in HCT-15cells.It seemed that the anthocyanidins contained in the grape rinds and red rice were eﬀective in the suppression of cell growth.Koide et al.(1997)also reported the anti-tumor eﬀects in vitro and in vivo of extracts from red soybeans,which were composed of mostly cyanin conjugated with glu-cose and rhamnose.Meanwhile,Gracia et al.(1997) reported that anthocyanins acted as antioxidants on human low-density lipoprotein(LDL)and lecithin-liposome systems in vitro.They found that the inhibi-tion of oxidation increased with the concentration of the antioxidant.In the LDL system,when the oxidation was catalyzed with10m M copper,malvidin was the best oxidation inhibitor,followed by delphinidin,cyanidin, and pelargonidin.When the oxidation was catalyzed with80m M copper,the order of antioxidant activity changed and decreased in the following order at all concentrations tested:delphinidin,cyanidin,malvidin, and pelargonidin.In the liposome system,catalyzed with either3or10m M copper,malvidin was the best inhibitor of both conjugated diene and hexanal forma-tion.At3m M copper,delphinidin,cyanidin,and pelar-gonidin showed prooxidant activity.At10m M copper, pelargonidin followed malvidin in antioxidant potency, and cyanidin and delphinidin were prooxidants.All the eﬀects of the anthocyanins listed above can be explained by several antioxidant mechanisms,includ-ing hydrogen donation,metal chelation,and protein binding.928J.-M.Kong/Phytochemistry64(2003)923–933。

高中英语第二部分 VOA慢速英语《美国万花筒》第20课(文本)素材

高中英语第二部分 VOA慢速英语《美国万花筒》第20课（文本）素材英语翻议讲解：1.abolitionist n.废除主义者,废奴主义者2.arsenal n.兵工厂,军械库,武器，军火库3.capture vt.抓取,获得,迷住例句：A large reward is offered for the capture of the criminals. 巨额悬赏捉拿这些罪犯。

The novel captured the imagination of thousands of readers. 这部小说引起了千万读者的想象。

4.sustainable adj.(对自然资源和能源的利用)不破坏生态平衡的, 合理利用的可持续的例句：Water Resource Protection and Sustainable Utilization 水资源保护与可持续利用。

1.To some people this building is the scene of a crime, where a murderer and a traitor was captured and brought to justice.bring to justice使归案受审例句：The police must do all they can to bring the criminals to justice. 警方必须尽力把罪犯送交法庭审判。

2.She is using her fame as a race car driver to bring environmental issues to the attention of millions of racing fans.bring to the attention of引起某人的注意例句：That way, you can bring a pattern of behavior to management's attention. 这样你就可以提请公司主管注意你老板的不当行为。

Vitis vinifera L. cv. Graciano grapes characterized by its anthocyanin profile.

Postharvest Biology and Technology 31(2004)69–79Vitis vinifera L.cv.Graciano grapes characterized by itsanthocyanin proﬁleV .Núñez,M.Monagas,M.C.Gomez-Cordovés ∗,B.BartoloméInstituto de Fermentaciones Industriales,CSIC,Juan de la Cierva 3,Madrid 28006,SpainReceived 20June 2002;accepted 30June 2003AbstractGraciano is a Spanish Vitis vinifera L.variety traditionally used to improve wine mixtures containing other original vari-eties.Given the scant literature on anthocyanins in Graciano grapes,the aim of this work was to determine its anthocyanin composition on the basis of HPLC/MS proﬁles and to compare it to those of other well-known varieties such as Tempranillo and Cabernet-Sauvignon.Thanks to this technique,the isomer cis of malvidin-3-(6-p -coumaroyl)-glucoside has been identiﬁed in the three varieties,being the ﬁrst time this compound is reported in Vitis varieties.Anthocyanins in Graciano show a high proportion of peonidins,with peonidin-3-glucoside/malvidin-3-glucoside (PnG/MG)being considered as a potential marker for the characterization of the variety.However,the ratio between the sums of the p -coumaroyl/acetylated anthocyanins ( Cm/ Ac)allows the three varieties to be best distinguished,Tempranillo having the highest values followed by Graciano (intermediate)and Cabernet-Sauvignon (values of less than one).©2003Elsevier B.V .All rights reserved.Keywords:Graciano;Anthocyanins;HPLC/MS;Malvidin-3-(6-p -coumaroyl)-glucoside isomer cis1.IntroductionAnthocyanins are the main agents responsible for the color of red grapes and the wines produced from them.They are located in the skin of the grapes (in the vacuoles of the ﬁrst three or four cell layers of the hypodermis),and are made during véraison.In Vitis vinifera L.,anthocyanins are monoglucosides,made up of an anthocyanidin [malvidin (M),delphinidin (D),peonidin (Pn),petunidin (Pt)or cyanidin (Cy)]∗Corresponding author.Tel.:+34-91-5622900;fax:+34-91-5644853.E-mail address:cgcordoves@iﬁ.csic.es (M.C.Gomez-Cordov´e s).substituted at position 3by a molecule of glucose.The glucosides,in turn,can be acylated at position 6of the sugar,with either acetic,p -coumaric or caffeic acid.The concentration and proﬁle of anthocyanins in red grapes varies with species,variety,maturity,sea-sonal conditions,production area,viticultural practice and yield (Mazza,1995;Keller and Hrazdina,1998;Esteban et al.,2001;Vivas de Gaulejac et al.,2001).Anthocyanin composition has been studied by many authors in order to characterize the variety or establish the origin of grapes,using either spec-trophotometric methods to measure the global phe-nolic family content (Gonzalez-Sanjoséand Diez,1993)or HPLC to look at the anthocyanin proﬁle (Piergiovanni and V olonterio,1980;Hebrero et al.,0925-5214/$–see front matter ©2003Elsevier B.V .All rights reserved.doi:10.1016/S0925-5214(03)00140-670V.N´uñez et al./Postharvest Biology and Technology31(2004)69–791988;Baldi et al.,1993;Ortega Meder et al.,1994;Lanaridis and Bena-Tzourou,1997;Mazza et al.,1999;Roggero et al.,1986a;Flamini and Tomasi,2000;Gonzalez-Neves et al.,2001).Some authorsbased their work on the relative abundance of individ-ual anthocyanin-3-glucosides.Roggero et al.(1986a)used the relationship between malvidin-3-glucosideand peonidin-3-glucoside(MG/PnG)contents,whileOrtega Mender et al.(1994)looked at the malvidin-3-glucoside/delphinidin-3-glucoside(MG/DG)ratio.Others established relationships between the to-tals of the different anthocyanidins.Baldi et al.(1993)analyzed the concentrations of derivativesof delphinidin,petunidin and malvidin with re-spect to those of peonidin( D/ Pn, Pt/ Pn and M/ Pn),while Roggero et al.(1988)investigated the ratio of the sum of the malvidin,petunidinand delphinidin contents and that of the derivativesof peonidin( M+ Pt+ D/ Pn).These pa-rameters are related to the enzymatic activities ofﬂavonoid-3 -hydroxydase(which transforms cyanidininto delphinidin and might turn peonidin into petuni-din)and o-dihydroxyphenyl-o-methyltransferase(re-sponsible for the methylation that occurs at positions3 and5 ,inducing the transformation of cyanidin intopeonidin,delphinidin into petunidin,and petunidininto malvidin)(Ortega Meder et al.,1994;Roggeroet al.,1986b).Another widely used classiﬁcation is based on thepresence and relative abundance of anthocyanins acy-lated with acetic(Ac)and p-coumaric(Cm)acids(Gonzalez-Sanjoséand Diez,1993;Ortega Mederet al.,1994;Hmamouchi et al.,1995).Marx et al.(2000),using the ratio PnAc+MAc/PnCm+MCm,and Gómez-Cordovés(1998),by means of summationof the acetyl/p-coumaroyl derivatives( Cm/ Ac),established these relationships as markers of variety.This classiﬁcation is therefore related to the grape’sacetyl and cinamyl transferase activity(Roggero et al.,1988).The production of each of these enzymes de-pends on the variety since they are direct expressionsof the genome.The Graciano grape is a red variety originally fromthe La Rioja and Navarra regions of Spain.DNA anal-ysis(Ibañez et al.,1999)has shown that the vari-eties Graciano,Parraleta de Somontano and Tintilla deRota(Spain),and Morrastel(France)are in fact oneand the same.Ampelographic analysis has also led to the conclusion that Miúda(Portugal),and Bastardo Nero,Bordelais,Cagnolate,Cagnovali,Nero,Cagnu-lari,Cagnulari Bastardo,Cagnulari Sardo,Caldare-dou,Caldarello,Cargo Muol,Couthurier,Graciano Tinto,Gros Negrette,Minustello,Tinta do Padre Anto-nio,Tinta Miuda,Xerez and Zinzillosa(Spain)are all the same variety too.Graciano,Parraleta de Somon-tano and Tintilla de Rota belong to the same caste. Few Spanish vineyards grow Graciano(0.02%).In the major wine Spanish region of La Rioja,Graciano makes up0.7%of the grape harvest,while in Navarra it forms0.6%.In recent years its cultivation has extended to other Spanish regions,Extremadura and Castilla-La Mancha.In France it is more widely grown.Graciano is a low yielding variety but it shows more resistance to disease(it is not very sensitive to oidium and spi-dermites,but is sensitive to botrytis and very sensitive to mildew)and bad weather(frosts and wind cause little damage but it is sensitive to drought and hail because of its medium consistency)than other vari-eties such as Tempranillo.The strain grows well on clayey-salty soils.According to the Riojan Regulatory Council,“Wines made with the Graciano variety are fresh.It is the most aromatic of all varieties used in this appellation controlléregion,has an important acidity and an intense red color.”It is traditionally used to improve mixtures containing Tempranillo,Garnacha and Mazuelo,giving them more aroma,and a more re-ﬁned quality,although it can provide both young and oak-aged monovarietal wines of great quality since it has excellent aging possibilities(Cirami et al.,2000). Given the increasing interest in the Graciano aro-matic and color attributes and the scant literature on this matter(Gonzalez-Sanjoséand Diez,1993),the aim of this work was to determine the anthocyanin composition of Graciano grape skins on the basis of HPLC/MS proﬁles.The results are compared to those of other varieties such as Tempranillo,a well known Spanish variety,and Cabernet-Sauvignon,which is an international grown French original variety.2.Materials and methods2.1.Plant materialSamples were collected from three vineyards of V. vinifera varieties Graciano,Cabernet-Sauvignon andV.N´uñez et al./Postharvest Biology and Technology31(2004)69–7971Tempranillo,cultivated at the Viticulture and Enol-ogy Station of Navarra(EVENA,Spain).Grapes werefrom two vintages(2000and2001)and were harvestedat their technological maturity,which wasﬁxed ac-cording to the established vintage schedule in the area.For quantitative analysis,two groups of100berrieseach randomly selected from all samples were col-lected for each grape variety.2.2.Preparation of grape skinsThe grapes were peeled manually and the skinswere removed from the rest of the grape.Skins werelyophilized and frozen immediately at−18◦C undernitrogen for subsequent analysis.Anthocyanin extracts were prepared by macerationof2.5g of milled skins in50ml of methanol–formicacid(95:5,v/v)for12h,the coloured liquid was sep-arated from the solid matrix and replaced with freshsolvent twice.The three combined solutions were con-centrated in a rotary evaporator up to25ml,avoidingtemperatures higher than35◦C.Distilled water wasadded to the sample up to50ml and kept at−18◦Cunder nitrogen until analysis.Each extraction proce-dure was carried out in duplicate.2.3.Families of phenolic compounds bycolorimetric analysisExtracts from grape skins were assayed for:totalpolyphenols(TP)(Singleton and Rossi,1965),proan-thocyanidins(PRO)(Riberau-Gayon and Stonestreet,1966),catechins(CAT)(Swain and Hillis,1959)and total anthocyanins(TA)(Paronetto,1977).Totalpolyphenols were expressed as gallic acid on a driedskin basis(g kg−1),proanthocyanidins as cyanidinchloride on a dried skin basis(g kg−1),catechins as d-(+)-catechin on a dried skin basis(g kg−1),and total anthocyanins as malvidin-3-glucoside on a driedskin basis(g kg−1).Every analysis was carried out induplicate.2.4.Analysis by liquid chromatographyA Waters(Milford,MA)HPLC chomatographequipped with a600-MS controller,a717plus au-tosampler,and a996photodiode-array detector wasused.A gradient of solvent A(water/formic acid,90:10,v/v)and solvent B(water/methanol/formic acid,45:45:10,v/v/v)was applied to a reversed-phase Nova-pack C18column(150mm×3.9mm)as fol-lows:15–80%B linear(0.8ml/min)from0to30min, 80%B isocratic(0.8ml/min)from30to43min and washing(100%methanol)and re-equilibration of the column from43to75min.Detection was performed by scanning from260to600nm.Quantiﬁcation was carried by external standard at530nm and expressed as malvidin-3-glucoside chloride on a dried skin ba-sis(g kg−1).Grape skin extracts(40␮l for samples of vintage2000and20␮l for samples of vintage 2001),previouslyﬁltered through0.45␮m,were in-jected into the HPLC.Injections were carried out in duplicate.2.5.Liquid chromatography/electrospray mass spectrometry(LC/ESI-MS)A Hewlet-Packard series1100(Palo Alto,CA) chromatograph equipped with DAD and MS detectors was used.Chromatographic and separation conditions were the same as reported above.Nitrogen was used as the nebulizing and drying gas.Operation was in positive mode and ESI conditions were as follows: nitrogen pressure,379kPa;drying gas,10ml/min at 350◦C;ion spray voltage,4000V;and fragmentor voltage,100V from0to24min and120V from24 to55min.3.Results and discussion3.1.Phenolic contentThe total polyphenol(TP)contents of the grape skins of Graciano and Tempranillo were similar.Both were greater than in Cabernet-Sauvignon variety in both study years(Table1).However,the total antho-cyanin(TA)was clearly greater in Graciano.These results agree with those of other authors who analyzed wines made from the same varieties(Almela et al., 1996;Arozarena et al.,2000).Catequin(CAT)values were greater for Tem-pranillo,followed by Cabernet-Sauvignon andﬁnally Graciano.Skins of the latter showed low concen-trations of these compounds,especially in the2000 harvest.The procyanidins(PRO)were found in72V.N´uñez et al./Postharvest Biology and Technology31(2004)69–79Table1Phenolic content in grape skin extracts from cv.Graciano,Tempranillo and Cabernet-SauvignonVariety Harvest TP TA CAT PRO Graciano200016.5±0.422.6±0.4<0.525.9±1.2 200129.9±2.542.9±2.7 4.3±0.244.5±3.7 Tempranillo200018.2±0.118.2±0.98.2±1.127.7±2.0 200129.1±1.826.9±1.612.3±0.847.2±1.1 Cabernet-S200011.4±1.216.1±0.7 1.8±0.217.0±1.6 200121.2±1.123.3±0.3 6.2±0.525.0±2.8∗Mean±S.D.(n=4).TP:total polyphenols(gallic acid on a dried skin basis,g kg−1);TA:total anthocyanins(malvidin-3-glucoside on a dried skin basis,g kg−1);CAT:catechins(d-(+)-catechin on a dried skin basis,g kg−1);PRO:proanthocyanidins(cyanidin chloride on a dried skin basis,g kg−1).greater quantity in Graciano and Tempranillo than in Cabernet-Sauvignon.The different phenolic contents of the skins(TP, TA,CAT and PRO)were greater for all varieties in 2001than in2000.This variation is attributable to the different weather of the two years.3.2.Individual anthocyaninsQualitative and quantitative differences were found in the anthocyanin proﬁles for the skins of the three varieties(Fig.1),which were very similar for the two study years.However,the chromatographic response (Abs530nm)was greater for the grape skins of2001 in all varieties.The same was seen for phenolic content (Table1).HPLC/MS identiﬁed the following anthocyanins in Graciano,Tempranillo and Cabernet-Sauvignon (Table2):(a)3-Glucosides of delphinidin(DG)(1),cyanidin(CyG)(2),petunidin(PtG)(3),peonidin(PnG)(5)and malvidin(MG)(6).(b)3-(6-Acetyl)-glucosides of delphinidin(DAc)(7),cyanidin(CyAc)(9),petunidin(PtAc)(11),pe-onidin(PnAc)(12)and malvidin(MAc)(14). (c)3-(6-p-Coumaroyl)-glucosides of delphinidin(DCm)(13),cyanidin(CyCm)(17),petunidin (PtCm)(19),peonidin(PnCm)(20)and malvidin (MCm)(21).(d)3-(6-Caffeoyl)-glucosides of peonidin(PnCf)(15)and malvidin(MCf)(16).An unknown peak(18)was also found with the same molecular mass and fragmentation as malvidin-3-(6-p-coumaroyl)-glucoside(22),but which eluted three minutes earlier in the chromatogram.Its UV spectrum was also slightly different(Fig.2),with absorption maxima at538and280nm(the latter with slightly greater amplitude)compared to535and 283nm for malvidin-3-(6-p-coumaroyl)-glucoside.To test whether they were isomers,extracts of the three varieties of the2001harvest were exposed to UV light (254nm for6h).The area of the unknown peak(18) increased1361%in Graciano,1089%in Tempranillo and1035%in Cabernet-Sauvignon,while the area cor-responding to malvidin-3-(6-p-coumaroyl)-glucoside (21)fell81%in Graciano,69%in Tempranillo and51%in Cabernet-Sauvignon,and the area of the remaining anthocyanins were less changed (75–93%).These data indicated that the unknown peak(18)corresponds to a cis form of malvidin-3-(6-p-coumaroyl)-glucoside.Peak21corresponds to the trans form.The cis isomer of malvidin-3-(6-p-couma-royl)-glucoside(18)was found in all three varieties studied,and in quantities that could be measured.A mass signal also appeared for the cis isomer of peonidin-3-(6-p-coumaroyl)-glucoside in Gra-ciano and Tempranillo,but it could not be quanti-ﬁing the area values for the two isomers in the mass chromatograms,the percentage of trans malvidin-3-(6-p-coumaroyl)-glucoside was calculated with respect to the cis form.Theﬁgures were similar for all three varieties:Graciano(10.2%),Tempranillo (11.9%)and Cabernet-Sauvignon(11.2%)(2001har-vest).No references reporting these compounds in Vitis varieties were found,though other authors have found isomers of anthocyanins(malvidin and del-phinidin)steriﬁed with p-coumaric acid in PetuniaV.N´uñez et al./Postharvest Biology and Technology31(2004)69–7973Fig.1.HPLC chromatograms of grape skin extracts recorded at530nm from Graciano,Tempranillo and Cabernet-Sauvignon.Peak identiﬁcation is shown in Table2.74V .N´u ñez et al./Postharvest Biology and Technology 31(2004)69–79Table 2Anthocyanin compounds detected in grape skin extracts from Graciano,Tempranillo and Cabernet-Sauvignon Peak poundt R M +m /z λmax GracianoCabernet-STempranillo1Delphinidin-3-glucoside 6.5465303525∗∗∗2Cyanidin-3-glucoside 8.5449287518∗∗∗3Petunidin-3-glucoside 10.3479317526∗∗∗4Unknown11.8657331530∗∗∗5Peonidin-3-glucoside 12.3463301518∗∗∗6Malvidin-3-glucoside13.4493331526∗∗∗7Delphinidin-3-(6-acetyl)-glucoside 16.7507303527∗∗∗8Unknown16.7517355528∗∗∗9Cyanidin-3-(6-acetyl)-glucoside 19.3491287519∗∗∗10Unknown19.9547385533n.d.∗∗11Petunidin-3-(6-acetyl)-glucoside 20.852*******∗∗∗12Peonidin-3-(6-acetyl)-glucoside23.2505301520∗∗∗13Delphinidin-3-(6-p -coumaroyl)-glucoside 23.6611303532∗n.d.∗14Malvidin-3-(6-acetyl)-glucoside 24.1535331531∗∗∗15Peonidin-3-(6-p -caffeoyl)-glucoside 25.1625301522∗∗∗16Malvidin-3-(6-p -caffeoyl)-glucoside 25.6655331535∗∗∗17Cianidin-3-(6-p -coumaroyl)-glucoside 26.1595287528∗∗∗18Malvidin-3-(6-p -coumaroyl)-glucoside cis 26.5639331538∗∗∗19Petunidin-3-(6-p -coumaroyl)-glucoside 27.1625317533∗∗∗20Peonidin-3-(6-p -coumaroyl)-glucoside29.4609301524∗∗∗21Malvidin-3-(6-p -coumaroyl)-glucoside trans29.7639331535∗∗∗t R :retention time;∗:detected;n.d.:not detected.a u0.000.050.100.150.200.250.300.350.40nm283535280538Fig.2.UV-Vis spectra of malvidin-3-(6-coumaroyl)-glucoside cis (---)and malvidin-3-(6-p -coumaroyl)-glucoside trans (—).V.N´uñez et al./Postharvest Biology and Technology31(2004)69–7975integrifolia and Triteleia bridgesii(George et al., 2001).Among the unknown compounds,two appear,one before and one after cyanidin-3-(6-acetyl)-glucoside (8,10),with molecular ion(M+)and fragmentation (m/z)values corresponding to cyanidin-3-glucoside pyruvate and petunidin-3-glucoside pyruvate,respec-tively.However,they do not correspond to the elution orders reported by other authors(Revilla et al.,1999). In order to compare the anthocyanin proﬁle among varieties,in the present study relationships were made between the different individual anthocyanin concentrations and that of malvidin-3-glucoside since the latter is the most abundant in the skins of Gra-ciano,Tempranillo and Cabernet-S and indeed in all V.vinifera.L.varieties(Mazza,1995)(Table3).In this way,the data can be normalized(since they come from different harvests)and their analysis simpli-ﬁed.The malvidin-3-glucoside content was greatest in Graciano(8.35and18.26malvidin-3-glucoside on a dried skin basis(g kg−1)in2000and2001, respectively),followed by Tempranillo(7.18and11.51malvidin-3-glucoside on on a dried skin basis(g kg−1))and Cabernet-Sauvignon(6.70and8.18 malvidin-3-glucoside on a dried skin basis(g kg−1)). These values agree with those for the total antho-cyanins described above(Table1).3.3.Simple glucosidesGraciano showed a high peonidin-3-glucoside con-tent in comparison to malvidin-3-glucoside(PnG/MG) (Table3).This could serve to distinguish this variety from Tempranillo and Cabernet-Sauvignon.Peonidin-3-glucoside is the second most abundant pigment in Graciano after malvidin-3-glucoside.Tempranillo showed a slightly greater amount of delphinidin-3-glucoside and signiﬁcantly more petunidin-3-glucoside with respect to its malvidin-3-glucoside content (DG/MG and Pt/MG)than did the other varieties studied(Table3).Calculating these same relation-ships of the different varieties from the anthocyanin contents published by other authors(Hebrero et al., 1988;Cacho et al.,1992;Ortega Meder et al., 1994;Mazza,1995;Lanaridis and Bena-Tzourou, 1997;Wulf and Nagel,1978;Yokotsuka et al., 1999;Esteban et al.,2001;Vivas de Gaulejac et al., 2001),the ratios DG/MG and PtG/MG were,in the majority of cases,greater in Tempranillo than in Cabernet-Sauvignon.They are also greater in Tem-pranillo than in Syrah(Mazza,1995),Merlot Noir, Cabernet-Franc(Vivas de Gaulejac et al.,2001),red Garnacha,Juan Garcia and Mencia(Ortega Meder et al.,1994).The cyanidin-3-glucoside content with respect to malvidin-3-glucoside(CyG/MG)was sim-ilar in all three varieties.The relationship is almost constant for all the derivatives of cyanidin,which would appear to indicate that this anthocyanidin is the base molecule from which the rest are formed, and that its lower content is owed to an equilibrium in this direction because of the enzymes mentioned earlier(see Section1).3.4.Acetyl derivativesAccording to some authors(Gonzalez-Sanjoséand Diez,1993;Ortega Meder et al.,1994;Gonzalez-Neves et al.,2001),the percentage of acylated anthocyanins, and in particular those in the form of acetates,are those that most contribute to varietal differences.Values for delphinidin-3-(6-acetyl)-glucoside,petunidin-3-(6-ac-etyl)-glucoside,and malvidin-3-(6-acetyl)-glucoside with respect to malvidin-3-glucoside(DAc/MG, PtAc/MG and MAc/MG)were greater in Cabernet-Sauvignon(Table3).Wulf and Nagel(1978)and Hebrero et al.(1988)provide data for these three compounds in Tempranillo and Cabernet-S.The DAc/ MG,PtAc/MG and MAc/MG ratios of Cabernet-Sauvignon are the highest.In agreement with that reported by Wulf and Nagel(1978),malvidin-3-(6-acetyl)-glucoside was the second most abundant pigment in Cabernet-Sauvignon after malvidin-3-glu-coside.With respect to malvidin-3-glucoside,the rest of the acetylated anthocyanins have a similar ratio in all three varieties.3.5.p-Coumaroyl derivativesMalvidin-3-(6-p-coumaroyl)-glucoside is consid-ered,along with malvidin-3-(6-acetyl)-glucoside,to be one of the most important derivatives for the char-acterization of varieties(Mazza,1995;Gonzalez-Sanjoséand Diez,1993;Lanaridis and Bena-Tzourou, 1997).Graciano had the greatest peonidin-3-(6-p-cou-maroyl)-glucoside content in both harvests.However, the proportion of this relative to malvidin-3-glucosideV .N´u ñez et al./Postharvest Biology and Technology 31(2004)69–7977(PnCm/MG)was not signiﬁcantly different to thatrecorded for the other two varieties (Table 3).Tem-pranillo showed the greatest amounts of delphinidin-3-(6-p -coumaroyl)-glucoside,petunidin-3-(6-p -couma-royl)-glucoside and malvidin-3-(6-p -coumaroyl)-glu-coside with respect to malvidin-3-glucoside (DCm/MG,PtCm/MG and MCm/MG).Malvidin-3-(6-p -coumaroyl)-glucoside was the most abundant pigment after malvidin-3-glucoside.The amount of cyanidin-3-(6-p -coumaroyl)-glucoside compared to malvidin-3-glucoside (CyCm/MG)was similar in all three varieties,as occurred for cyanidin-3-(6-acetyl)-gluco-side and cyanidin-3-glucoside.Delphinidin-3-(6-p -coumaroyl)-glucoside was not found in Cabernet-Sauvignon in either year,in contrast to that reported by other authors (Wulf and Nagel,1978;Revilla et al.,1999).3.6.Caffeoyl derivativesPeonidin-3-(6-caffeoyl)-glucoside is a compound that does not often appear in the literature sinceit Fig.3.Anthocyanin distribution by anthocyanidin and acylation in grape skin extracts from Graciano,Tempranillo and Cabernet-Sauvignon.tends to be present in small quantities in particular varieties and because it is difﬁcult to identify by its UV spectrum alone (Hebrero et al.,1988).It was ﬁrst identiﬁed by Piergiovanni and V olonterio (1980)in ter,Hebrero et al.(1988),discovered its presence in Tempranillo grapes,Mazza et al.(1999)recorded it in Merlot and Cabernet Franc,Baldi et al.(1993)in Colorino Nipozzano,and Mateus et al.(2001)reported it in Touriga Nacional and Touriga Francesa.In the present work,HPLC/MS identiﬁed it in all three studied varieties.Graciano showed the highest content in each year.However,when com-pared to the malvidin-3-glucoside content,the ratio (PnCf/MG)was similar in all three varieties (Table 3).The ratio of malvidin-3-(6-caffeoyl)-glucoside to its precursor (MCf/MG)was also similar in all three varieties.According to Gonzalez-Sanjoséand Diez (1993),this compound,along with the rest of the mal-vidin derivatives,provides important discriminating power.However,this was not the case in the present work since concentrations varied widely between the two harvests.78V.N´uñez et al./Postharvest Biology and Technology31(2004)69–793.7.Anthocyanin distribution by anthocyanidin and acylation derivativesFig.3shows the distribution of the anthocyanins in the skins of the three varieties(2001harvest)grouped by the following:(a)anthocyanidin:delphinidins(DG+DAc+DCm),cyanidins(CyG+CyAc+CyCm), petunidins(PtG+PtAc+PtCm),peonidins (PnG+PnAc+PnCm+PnCf)and malvidins (MG+MAc+MCm+MCf);(b)acylation:non-acylated glucosides(DG+CyG+PtG+PnG+MG),acetyl derivatives(DAc+ CyAc+PtAc+PnAc+MAc),p-coumaroyl deriva-tives(DCm+CyCm+PtCm+PnCm+MCm) and caffeoyl derivatives(PnCf+MCf).The different varieties showed different antho-cyanidin distributions.The percentage of malvidins was similar in all three varieties,and the highest of all the anthocyanidins.This is logical if one bears in mind that malvidin is the terminal anthocyanidin in biosynthesis of anthocyanins(Ortega Meder et al., 1994).The second most abundant anthocyanidin in Graciano was peonidin.This was not seen in the other two varieties where delphinidin and petunidin were the most mon to all varieties are low concentrations of cyanidins—which is to be expected since this anthocyanin is the precursor of all others(Ortega Meder et al.,1994;Roggero et al., 1986b).By acylation,the non-acylated glucoside group were the most abundant in the three varieties studied, with Graciano showing the greatest percentage of these derivatives.Acetyl derivatives were very abun-dant in Cabernet-Sauvignon,while the p-coumaroyl derivatives were the most abundant in Tempranillo. This has already been reported by Hebrero et al. (1988)who analyzed Tempranillo and compared results with those of Wulf and Nagel(1978)in Cabernet-Sauvignon.With regard to caffeoyl deriva-tives,the percentage was very similar,and very low in the three varieties studied.If the sum of the p-coumaroyl derivatives is divided by the sum of the acetyl derivatives( Cm/ Ac),val-ues of1.9and2.7are obtained for Graciano for the 2000and2001harvests,respectively,5.5and9.4for Tempranillo,and0.3and0.3for Cabernet-Sauvignon.This allows the three varieties to be distinguished and characterized.4.ConclusionsThis paper shows signiﬁcant quantitative differ-ences in the anthocyanin composition of skins from Graciano,Tempranillo and Cabernet-Sauvignon. When using HPLC/MS,a peak was discovered corre-sponding to the cis isomer of malvidin-3-(6-p-coumar-oyl)-glucoside,a compound never before reported in Vitis.Peonidin-3-(6-caffeoyl)-glucoside was also found in greatest quantity in Graciano.This variety is characterized by having a high concentration of peonidin derivatives.This is shown by the high peo-nidin-3-glucoside/malvidin-3-glucoside ratio,which could serve as a marker for the characterization of the variety.Graciano has a higher content in simple an-thocyanin glucosides than Tempranillo or Cabernet-Sauvignon.The ratio between the sums of the p-coumaroyl/acetylated anthocyanins allows the three varieties to be distinguished,Tempranillo having the highest value followed by Graciano(intermediate) and Cabernet-Sauvignon(values of less than one). However,more studies with grapes from other loca-tions and cultivar conditions should be carried out in order to conﬁrm these observations. AcknowledgementsThe authors are grateful to Mr.Julian Suberviola (EVENA,Navarra,Spain)for providing the grape samples,to Ms.Isabel Izquierdo for technical as-sistance in the HPLC analysis and to Mrs.Isabel Jimenez for technical assistance in the MS analysis. The authors also thank the Ministerio de Ciencia y Tecnolog´ıa for a F.P.I.predoctoral scholarship to V.N.,and the Spanish Comisión Interministerial de Ciencia y Tecnolog´ıa(CICYT)for funding(Project AGL2000-1427-C02-02).ReferencesAlmela,L.,Javaloy,S.,Fernández-Lopez,J.A.,López-Roca,J.M., 1996.Varietal classiﬁcation of young red wines in terms ofV.N´uñez et al./Postharvest Biology and Technology31(2004)69–7979chemical and colour parameters.J.Sci.Food Agric.70,173–180.Arozarena,I.,Casp, A.,Mar´ın,R.,Navarro,M.,2000.Multivariante differentiation of Spanish red wines according to region and variety.J.Sci.Food Agric.80,1909–1917. Baldi, A.,Romani, A.,Mulinacci,N.,Vincieri, F.F.,1993.Composés phenoliques dans les cépages de Toscane de Vitis vinifera L.J.Int.Sci.Vigne Vin.27,201–215.Cacho,J.,Fernandez,V.Ferreira,Castells,J.E.,1992.Evolution of ﬁve anthocyanidin-3-glucosides in the skin of the Tempranillo, Moristel and Garnacha grape varieties and inﬂuence of climatological variables.Am.J.Enol.Vitic.43,244–248. Cirami,R.,Ewart,A.,Furkaliev,J.,2000.The viticultural and oenological evaluation of Graciano,Gramon and Harslevelu.The Australia Grapegrower&Winemaker436,20–23. Esteban,M.A.,Villanueva,M.J.,Lissarrague,J.R.,2001.Effect of irrigation on changes in the anthocyanin composition of the skin of cv Tempranillo(Vitis vinifera L.)grape berries during ripening.J.Sci.Food Agric.81,409–420.Flamini,R.,Tomasi,D.,2000.The anthocyanin content in berries of the hybrid grape cultivars Clinton and Isabella.Vitis39, 79–81.George, F.,Figueiredo,P.,Toki,K.,Tatsuzawa, F.,Saito,N., Brouillard,R.,2001.Inﬂuence of trans-cis isomerisation of coumaric acid substituents on colour varience and stabilisation in anthocyanins.Phytochemistry57,791–795.Gonzalez-Neves,G.,Gomez-Cordovés, C.,Barreiro,L.,2001.Anthocyanic composition of Tanta,Cabernet Sauvignon and Merlot Young red wines from Uruguay.J.Wine Res.12,125–133.Gonzalez-Sanjosé,M.L.,Diez,C.,1993.Caracterización varietal en función de la composición atociánica de la uva:análisis discriminante.Agrochimica XXXVII(1/2),86–92.Gómez-Cordovés,C.1998.Inﬂuencia de la variedad,el clon y la producción de la vid,sobre el perﬁl antociánico de vinos tintos.II-Oenologie.OIV II.pp.213–220.Hebrero,E.,Santos-Buelga,C.,Rivas-Gonzalo,J.C.,1988.High performance liquid chromatography diode array spectroscopy identiﬁcation of anthocyanins of Vitis vinifera variety Tempranillo.Am.J.Enol.Vitic.39,227–233. Hmamouchi,M.,Es-Saﬁ,N.,Pellecuer,J.,Essassi,E.M.,1995.Anthocyanic composition of grape skins of four red grape varieties grown in Morocco.Bulletin de L’O.I.V.777–778, 907–919.Ibañez,J.,de Andrés,M.T.y Borrego,J.1999.Identiﬁcación de las variedades de vid mediante análisis de ADN.Sevi.No.2776/77:3713–3720.Keller,M.,Hrazdina,G.,1998.Interaction of nitrogen availability during bloom and light intensity during verason.II.Effects on antocyanin and phenolic development during grape ripening.Am.J.Enol.Vitic.49,341–348.Lanaridis,P.,Bena-Tzourou,I.,1997.Study of anthocyanins’variations during the ripening ofﬁve vine red varieties cultivated in Greece.J.Int.Sci.Vigne Vin.31,205–212. Marx,R.,Holbach,B.,Otteneder,H.2000.Determination of nine characteristic Anthocyanins in wine by HPLC.F.V.No.1104 OIV.Mateus,N.,Proença,S.,Ribeiro,P.,Machado,J.M.,De Freitas,V., 2001.Grape and wine polyphenolic composition of red Vitis vinifera varieties concerning vineyard altitude.Cienc.Technol.Aliment.3,102–110.Mazza,G.,1995.Anthocyanins in grapes and grape products.Crit.Rev.Food Sci.Nutr.35,341–371.Mazza,G.,Fukumoto,L.,Delaquis,P.,Girard,B.,Ewert,B., 1999.Anthocyanins,phenolics,and color of Cabernet Franc, Merlot,and Pinot Noir wines from British Columbia.J.Agric.Food Chem.47,4009–4017.Ortega Meder,M.D.,Rivas Gonzalo,J.C.,Vicente,J.L.,Santos Buelga,C.,1994.Differentiation of grapes according to the skin anthocyanin composition.Revista Española de Ciencia y Tecnolog´ıa de Alimentos.34,409–426.Paronetto,L.,1977.Polifenoli e Tecnica Enologica.Selepress, Milan,Italy.Piergiovanni,L.,V olonterio,G.,1980.Tecniche cromatograﬁche nello studio della frazione antocianica dell uve.Riv.Vit.Enol.33,289–309.Revilla,I.,Perez-Magariño,S.,González-SanJosé,M.L.,Beltrán, S.,1999.Identiﬁcation of anthocyanin derivatives in grape skin extracts and red wine by liquid chromatography with diode array and mass spectrometric detection.J.Chromatogr.A.847, 83–90.Riberau-Gayon,P.,Stonestreet,E.,1966.Dósage des tannins du vin rouges et determination du leur structure.Chem.Anal.48, 188–196.Roggero,J.P.,Coen,S.,Larice,J.L.,1986a.Etude comparative de la compositon anthocyanique des cepages.Essai de classiﬁcation.Bull.Liaison Gr.Polyphénols.13,380–388. Roggero,J.P.,Coen,S.,Ragonnet,B.,1986b.High performance liquid chromatography survey on changes in pigment content in ripening grapes of Syrah.An approach to anthocyanin metabolism.Am.J.Enol.Vitic.37,77–83.Roggero,J.P.,Larice,J.L.,Rocheville-Divore, C.,Archier,P., Coen,S.,position anthocyanique des cépages.II.Essai de classiﬁcaton sur trois ans par analyse en composantes principales et par analyse factorielle discriminante.Rev.Fr.Oenol.112,41–48.Singleton,V.I.,Rossi,J.A.,1965.Colorimetry of total phenolics with phosphomolibdicphosphotungstic acid reagent.Am.J.Enol.Vitic.16,144–158.Swain,T.,Hillis,W.E.,1959.The phenolic constituents of Prunus domestica.I.The quantitative analysis of phenolics constituents.J.Sci.Food Agric.10,63–69.Vivas de Gaulejac,N.,Nonier,M.F.,Guerra, C.,Vivas,N., 2001.Anthocyanin in grape skin during maturation of Vitis vinifera L.cv.Cabernet Sauignon and Merlot Noir from different Bordeaux Terroirs.J.Int.Sci.Vigne Vin.35,149–156.Wulf,L.,Nagel,C.W.,1978.High-pressure liquid chromatographic separation of anthocyanins of Vitis vinifera.Am.J.Enol.Vitic.29,42–49.Yokotsuka,K.,Nagao,A.,Nakazawa,K.,Sato,M.,1999.Changes in anthocyanin in berry skins of Merlot and Cabernet-S grapes grown in two soils modiﬁed with limestone or oyster shell versus a native soil over two years.Am.J.Enol.Vitic.5,1–12.。

苹果愈伤过表达MdMYB1对花色素苷合成的影响

苹果愈伤过表达MdMYB1对花色素苷合成的影响CHEN Li;YAO Yuncong【摘要】[目的]苹果生产是北京市都市型农业的支柱产业之一.果实色泽是苹果果实重要的外观品质,主要由花色素苷的含量决定.MdMYB1转录因子在调控花色素苷及类黄酮的生物合成中发挥着重要的作用.[方法]将MdMYB1转录因子在‘王林’(Malus domestica cv.‘Orin')愈伤组织进行过表达,进行持续光照和低温处理,观察愈伤组织类黄酮类物质变化,同时检测花色素苷生物合成基因表达水平的变化,确定其对花色素苷生物合成的调控功能.[结果]愈伤组织中过表达MdMYB1后,在持续光照和低温处理下花色素苷含量显著提高,并能够促进花色素苷生物合成基因的表达.[结论]该研究为深入了解MdMYB1调控花色素苷作用机理提供技术支撑.【期刊名称】《北京农学院学报》【年(卷),期】2019(034)003【总页数】5页(P37-41)【关键词】苹果;花色素苷;愈伤;转基因【作者】CHEN Li;YAO Yuncong【作者单位】;【正文语种】中文【中图分类】S661.4苹果为蔷薇科(Rosaceae)苹果属(Malus)落叶乔木，是世界四大水果之首[1]。

苹果产业在农业结构调整，农民增收和生态改良方面具有重要意义。

经过多年发展，目前中国苹果栽培面积、总产量和出口量均居世界首位[2-4]。

在苹果产量大幅提升的同时，苹果品质较低已经成为阻碍中国苹果产业发展的重要因素[5-7]。

苹果品质的提升对于苹果产业的发展显得尤为重要。

果实色泽是苹果果实重要的外观品质，能够在一定程度上影响其市场价格。

而红色果皮主要由花色素苷的含量决定[8]。

花色素苷生物合成的分子机理研究表明影响花色素苷生物合成的基因有两类：一类是不同植物共同具有的花色素苷生物合成基因，它们直接编码花色素苷生物合成酶类[9]；另一类是调节基因，调节花色素苷生物合成基因表达的强度和过程及色素在空间和时间上的积累[10]。

人教版全国全部高考专题英语高考真卷试卷及解析

人教版全国全部高考专题英语高考真卷1.阅读理解第1题.Scientists in Mexico say they may have found a way to cut the production of methane（甲烷）, a gas linked to rising temperatures on the Earth's surface. The scientists say their method may help reduce the methane released by cows, one of the main producers of the gas.When talking about global warming, many people think of carbon dioxide, another heat-trapping gas. However, methane is an even more powerful heat-trapping gas. Anyhow, cows are known to produce high levels of methane.Researchers at the Autonomous University of the State of Mexico are studying how a cow's diet affects the production of methane. The researchers are using a specially designed machine to measure the effect. The scientists use the machine to try to measure the animal's breathing to examine the methane released.Most of the gas is released when cows eat and process food. The digestive （助消化的）bacteria in a cow's stomach causes the animal to send out the gas through its mouth. There are an estimated 1.3 to 1.5 billion cows in the world. Each animal releases as much as 120 kilograms of methane per year. The U.S. Environmental Protection Agency reports that, whenever measured in pounds, the effect of methane on climate change is more than 25 times greater than carbon dioxide over a 100-year period.As it is difficult to capture the gas released by cows for use as energy, one way to lessen its release is to change their diet. Scientists in some countriesare looking for ways to decrease cow methane. But while they are trying different plants and chemical compounds, those products would be too costly and difficult to bring to Mexico.（1）How harmful is the gas sent off by cows?A: It is more harmful than carbon dioxide.B: It takes up most of human-caused methane.C: It is more difficult to control its amount.D: It is the key factor to prevent globe warming.（2）How is the harmful gas produced by cows?A: When their gas meets with air.B: When they breathe in air through mouths.C: When their gas is being sent out.D: When they digest food in their stomachs.（3）What can scientists in some countries do to settle the problem caused by cows?A: Use their gases as energy.B: Change what they eat.C: Limit the number of cows.D: Fix machines on their mouths.（4）What may be the best title for the text?A: Cows Are Much More Harmful than CarsB: We Had a Better Method of Raising CowsC: Two Gases Are Causing the Warming of the PlanetD: Researchers Found a Way to Reduce Methane from Cows【答案】ADBD【解答】（1）A 推理判断题。

过氧化亚硝酸阴离子的荧光检测

Visualizing Peroxynitrite Fluxes in Endothelial Cells Reveals the Dynamic Progression of Brain Vascular InjuryXin Li,†,∥Rong-Rong Tao,‡,∥Ling-Juan Hong,‡Juan Cheng,†Quan Jiang,‡Ying-Mei Lu,§Mei-Hua Liao,‡Wei-Feng Ye,‡Nan-Nan Lu,‡Feng Han,*,‡Yong-Zhou Hu,*,†and You-Hong Hu*,††ZJU-ENS Joint Laboratory of Medicinal Chemistry,College of Pharmaceutical Sciences,Zhejiang University,Hangzhou310058, China‡Institute of Pharmacology and Toxicology,College of Pharmaceutical Sciences,Zhejiang University,Hangzhou310058,China§School of Medicine,Zhejiang University City College,Hangzhou310015,China*Supporting Informationpharmacokinetic properties endow NP3with the capability to monitortemporal and spatial resolution.As a proof of concept,NP3hasformation in ischemia progression in live mouse brain by use of two-properties,NP3holds great promise for visualizing endogenousprogressions in vitro and in vivo.INTRODUCTIONPeroxynitrite(ONOO−),a highly reactive nitrogen speciesgenerated from the reaction between nitric oxide(NO)andsuperoxide(O2.‑)at a diﬀusion-limited rate of1.9×1010M−1·s−1under pathological conditions,1attracts increasing attentiondue to its“double-edged”character.2,3ONOO−may exert acontributory eﬀect by participating in nitrating tyrosinesignaling.4Nevertheless,ONOO−is more frequently regardedas deleterious due to its nitrosative damage to lipids,proteins,and DNA.5,6ONOO−has been implicated in various redox-related diseases,5,7,8including ischemia-reperfusion injury.9We have been particularly interested in the roles of ONOO−in the progression of brain ischemia-induced endothelial dysfunction and neurovascular pathogenic cascades.Our preliminary results have shown that ONOO−is formed under conditions of ischemia and that its nitrosative damage is implicated in neurovascular damage following cerebral ischemia.10−12Tradi-tional biological assay for ONOO−primarily relies on the immunostaining of3-nitrotyrosine.13This method has the major limitation of being incompatible with living systems and can therefore no longer satisfy research needs for tracking native ONOO−in real time with high spatial resolution,which is pivotal for fully understanding ONOO−pathology incontexts of ischemia.Fluorescence imaging employing small molecular probes, however,has emerged as a desirable and indispensable tool forinterrogating intact living samples.14,15Due to the obvioustechnical and practical advantages of good membranepermeability,high sensitivity,and operational simplicity,ﬂuorescence probes are attracting increasing attention in life scienceﬁelds,16−19especially two-photon excitable probes,because they are compatible with two-photonﬂuorescencemicroscopy and can therefore realize three-dimensionalimaging of biological specimens with deeper tissue penetration and less photodamage.20−22Indeed,severalﬂuorescent probes are commercially available to detect ONOO−,such as aminophenylﬂuorescein(APF)and hydroxyphenylﬂuorescein (HPF).23Unfortunately,these probes are limited by their poor selectivity for ONOO−against other highly reactive species, such as•OH or ClO−.24To address this problem,several research groups have set out to develop new probes,and thisReceived:July2,2015Published:September9,2015elegant work has resulted in the development of several selective probes suitable for imaging ONOO −in live cells,25−37live mice,30or even the redox cycles between ONOO −and glutathione.30,32,38Facilitated by these probes,visualization of O N O O −i n m a c r o p h a g e s d u r i n g i m m u n e r e -sponse 25−27,29,30,32,33,35or in mouse hearts under atheroscle-rosis 26has been realized.However,study on the real-time visualization of ONOO −production in the brain of live animals with ischemia-induced neurovascular damage is still lacking,which represents a great challenge due to the rigid require-ments for brain imaging agents including high speci ﬁcity,desired photophysical properties,and good blood −brain barrier (BBB)penetrability.39,40Herein,we report a two-photon ﬂuorescent “switch-on ”probe for the detection of ONOO −.The probe,judiciously designed by combining the basic principles of ﬂuorescent probe design and drug design,is highly speci ﬁc and sensitive toward ONOO −,two-photon excitable,and most importantly,readily BBB-penetrable.It is ﬂuorescence-silent in the absence of ONOO −but can respond rapidly to ONOO −with dramatic emission enhancement (utmost 600-fold).Its capability to track in situ generation of ONOO −in live cells and live mice with ischemia-induced neurovascular damage has been fully characterized.■RESULTS AND DISCUSSIONProbe Design and Synthesis.To image ONOO −in live animals,the ﬂuorophore selected for probe construction should be nontoxic,su ﬃciently bright,inert to other species in the complex biological context,excitable with deep-penetrating infrared light,and possess desired pharmacokinetic pro ﬁles.Among the prevalent ﬂuorescent markers for bioimaging,2-(2′-hydroxyphenyl)benzothiazole (BT)provoked our interest due to its druglike physical −chemical performance and good photophysical properties.First,the benzothiazole skeleton may exhibit the desired pharmacokinetic properties,especially the BBB penetration ability mandatory for brain imagingagents,as exempli ﬁed by [11C]PIB,an extensively studied positron emission tomography imaging probe for A βplaques in humans.41Second,the BT series of ﬂuorophores commonly exist in the normal form (N)with weak ﬂuorescence but can automerize under excitation (T)via a process called excited-state intramolecular proton transfer (ESIPT)(Figure 1),42accompanied by both enhancement and red shift of their ﬂuorescence.Blockage of this ESIPT e ﬀect with a special chemical motif that can react selectively with intended target in complex biological milieu to initiate the ESIPT process enables the design of sensitive ﬂuorescent probes.Third,BT ﬂuorophore is two-photon excitable and may be compatible with live tissue imaging.43With all these considerations,probe NP1was designed by blocking the ESIPT process with a saturated C −N bond (Figure 1).We envisioned that the great tendency to be aromatic would render NP1susceptible to oxidation by ONOO −and therefore restore the ESIPT process.NP1,as anticipated,could indeed respond to ONOO −with a swift ﬂuorescence intensity enhancement in phosphate-bu ﬀered saline (PBS;10mM,pH 7.4)(Figures S1and S2).However,NP1was found to be unstable when exposed to air.This instability challenges its selectivity.To develop probes with improved stability and speci ﬁcity,we next blocked the hydrogen donor of the ESIPT process and designed NP2by switching the hydroxyl group to N -methyl-p -hydroxyaniline,where the phenol group may be oxidized to benzoquinone by ONOO −,accompanied by N −C (sp 2)bond cleavage,thereby furnishing a proton donor (Figure 1).26NP3and NP4were also designed by installing electron-donating groups to the p -hydroxyaniline ring in order to make the hydroxyl group more susceptible to oxidants.All the probes were facilely synthesized via Mills reaction (Scheme S1).Probe Evaluation.We ﬁrst tested the ﬂuorescence responses of NP2−4toward ONOO −.These probes (5.0μM)alone were nearly nonemissive in PBS.In contrast,in the presence of ONOO −(10μM),all showed obvious ﬂuorescence enhancement,and NP3was the most sensitive one withanFigure 1.Structures of BT series of ONOO −probes and design philosophy.increase factor of 600-fold,followed by NP2and NP4(Figure S3).These results not only distinguish NP3as a desirable candidate for further study but also suggest that both electronic and steric e ﬀects should be considered for probe design because the steric e ﬀects of the methoxy groups adjacent to the hydroxyl group in NP4greatly outweigh their positive electron-donating e ﬀects.Spectroscopic Properties and Selectivity.To pro ﬁle the response of NP3toward ONOO −in detail,we examined its speci ﬁcity by recording its responses toward various reactive oxygen species (ROS)and reactive nitrogen species (RNS).Encouragingly,no analytes other than ONOO −could switch on the ﬂuorescence of NP3.NP3could still recognize ONOO −with a dramatic increase in ﬂuorescence intensity even in the presence of other reactive species,cations,or amino acids commonly found in biological systems,implying great speci ﬁcity of NP3toward ONOO −(Figure 2a and Figure S4).Next,the sensing kinetics was studied,and the reaction between NP3and ONOO −was found to be completed within seconds (Figure 2b and Figure S5),which is important given the extremely elusive nature of ONOO −.Moreover,the ﬂuorescence enhancement of NP3was linearly correlated with concentrations of ONOO −ranging from 0to 10μM,implying the great potential of NP3to quantify ONOO −(Figure 2c and Figure S6).The detection limit of NP3was as low as 5.0nM (S/N =3)(Figure S7).Notably,ONOO −detection by NP3was insusceptible to pH changes in the surroundings (Figure S8).Additionally,su ﬃcient photostability was observed for both NP3and the detection system (Figure S9),indicating the robustness of NP3.We also evaluated the ability of NP3to detect ONOO −in two-photon excitation mode by measuring ﬂuorescence spectra of the NP3−ONOO −mixture with two-photon excitation.As expected,sensitive signals remained.The two-photon absorp-tion cross sections (σ)of the system at 760−820nm were also determined with ﬂuorescein in H 2O (pH =13)as standard,44and σmax was observed at 820nm with a value of 3.6GM (Figure S10).These results ﬁrmly support the feasibility of NP3as a highly speci ﬁc and sensitive probe for ONOO −in vitro that shows promise for in vivo imaging.In agreement with the ﬂuorescence switch-on response,treating NP3(10μM)with ONOO −could also induce a dramatic change in its UV absorption pro ﬁle.NP3itself exhibited a major absorption band centered at 300nm (ε16310),whereas ONOO −treatment resulted in the disappear-ance of this band and the appearance of two new bands centered at 285and 350nm,both of which strengthened in a ONOO −concentration-dependent manner (Figure S11),indicating removal of the phenol ring and generation of an intramolecular hydrogen bond as shown in Figure 1.We also veri ﬁed the detection mechanism by isolating and characteriz-ing the reaction product by 1H NMR and HRMS spectral analysis (Figures S12and S13).45Determination of Plasma and Brain NP3Concen-trations.Pharmacokinetic study in C57mice showed that NP3readily crossed the blood −brain barrier (Figure S14).A 1.3-fold brain/plasma ratio was achieved after 0.5h of tail intravenous (iv)injection dosing at 10mg/kg.The absolute brain concentration reached a high level of 970ng/mL at 0.5h after NP3administration and only 136ng/mL remained after 2h,indicating the e ﬃcient brain penetration and fast brain clearance rate of NP3,which are deemed advantageous for brain imaging agents.Fluorescent Response of NP3to Dynamic Changes in Nitrosative Stress in Live Endothelial Cells.With the photophysical pro ﬁles of NP3fully characterized,we next investigated its feasibility for dynamically tracking intracellular ONOO −generation.A time-lapse series of single confocal plane images were taken to observe the NP3ﬂuorescence response toward ONOO −in live EA.hy926endothelial cells after incubation with or without 3-morpholinosydnonimine (SIN-1,0.5mM),an ONOO −donor.As shown in Figure 3,no detectable ﬂuorescence signal was observed in endothelial cells loaded with NP3in the absence of the ONOO −donor SIN-1(Figure 3a,b).However,upon exposure to SIN-1,intracellular ﬂuorescence in endothelial cells gradually increased 10min after SIN-1stimulation and kept increasing in a time-dependent manner until at least 50min after treatment (Figure 3a,b).In contrast,clearance of ONOO −with uric acid (100μM)or 5,10,15,20-tetrakis(4-sulfonatophenyl)porphyrinatoiron(III)chloride (FeTTPS;1μM)blunted the robustness of NP3ﬂuorescence in ONOO −-treated endothelial cells (Figure 3c and Figures S15and S16).NP3also worked well as an e ﬃcient ONOO −probe in other cell lines,as shown in human brain microvascular endothelial cells (HBMEC)in Figure S17.The ONOO −(Figure S17a )or SIN-1(Figure S17b )treatment-induced NP3ﬂuorescence elevation was also observed in a dose-dependent manner in HBMEC.These results indicate that NP3is applicable for real-time tracking of ONOO −generation in live cells and suggest its promise for serving as a molecular imaging tool to explore ONOO −biology under pathological conditions.Visualizing ONOO −Fluxes in Endothelial Cells after Oxygen −Glucose Deprivation.Considerable evidence has indicated that ONOO −overproduction in endothelial cells mediates cellular damage during cerebral ischemia.46−48Thus,it would be interesting if NP3could help to identify dynamic changes of ONOO −formation during endothelial ischemic injury.As shown in Figure 4a,time-dependent accumulationofFigure 2.Characterization of ﬂuorescent response of NP3toward ONOO −.(a)Fluorescent responses of NP3(5μM)toward various analytes (10μM).Data shown represent ﬂuorescent intensity at 470nm,30min after addition of various analytes.(b)ONOO −(ﬁnal concentration 10μM)was quickly injected into a solution of NP3(ﬁnal concentration 5μM),and the ﬂuorescent intensity at 470nm was plotted against time.(c)Fluorescence enhancement of NP3(5μM)at 470nm as a function of ONOO −(0−10μM)after 15min of reaction.All data were acquired in PBS (10mM,pH 7.4)with excitation at 375nm.NP3ﬂuorescence was observed in endothelial cells over 0.5−2h following oxygen −glucose deprivation (OGD)exposure.A similar pattern of ﬂuorescence was observed in HBMEC (Figure 4b).To elaborate the speci ﬁcity of NP3toward ONOO −during OGD treatment,FeTTPS (1μM),the ONOO −decomposition catalyst,blunted the elevation of NP3ﬂuorescence in OGD-treated endothelial cells (Figure 4c and Figures S18and S19),con ﬁrming that NP3is speci ﬁc for ONOO −during OGD insult.ONOO −-mediated stress can also be initiated by early intracellular Ca 2+release and calmodulin activation.49To discern whether the OGD-induced increase of NP3ﬂuorescence occurs in a Ca 2+/calmodulin-dependent manner,calmodulin inhibitors (W7and melatonin)and a Ca 2+-speci ﬁc aminopoly(carboxylic acid)[1,2-bis(o -aminophenoxy)ethane-N ,N ,N ′,N ′-tetraacetic acid,BAPTA]were used to bind Ca 2+/calmodulin signaling.As shown in Figure 4c and Figure S19,the OGD-induced increase of NP3ﬂuorescence was reduced in the presence of either calmodulin inhibitor or BAPTA.Taken together,these results suggest thatNP3is a highly selective and speci ﬁc probe for monitoring ONOO −ﬂuxes during ischemia.Real-Time Monitoring of Vascular Peroxynitrite Fluxes with High Temporal and Spatial Resolution in Live Cells.Mitochondria constitute a primary locus for intracellular ONOO −formation and targeting.50For example,modi ﬁcation of tyrosine residues by endogenous ONOO −results in inhibition of mitochondrial complex I.51To further visualize the subcellular distribution of ONOO −labeled by NP3ﬂuorescence,MitoRed (Invitrogen)was used to localize mitochondrial components in endothelial cells.NP3ﬂuores-cence in control endothelial cells was undetected (Figure 5and Figure S20).Interestingly,consistent with MitoRed local-ization,elevated NP3ﬂuorescence in endothelial cells after OGD was primarily observed to localize in the same components (Figure 5).Analysis of NP3ﬂuorescence (green)in mitochondrial components of endothelial cells revealed a signi ﬁcant elevation after 1h of OGD,and continuous elevation could be observed after 2h untilatFigure 3.Characterization of ONOO −formation by NP3in endothelial cells upon nitrosative stress.(a)Time-lapse series of single confocal plane images taken from living EA.hy926endothelial cells.The cells were seeded on glass-bottom 6-well plates overnight and then preincubated with NP3(5.0μM)for 30min,followed by stimulation with or without SIN-1(0.5mM).(b)Quantitative analysis of dynamic changes of NP3ﬂuorescence after SIN-1(0.5mM)treatment in panel a.Data are presented as a densitometric ratio change compared with control.(c)E ﬀects of ONOO −scavengers uric acid (100μM)and FeTTPS (1μM)on changes in NP3ﬂuorescence in endothelial cells in the presence of ONOO −(60μM).The ONOO −scavengers were preincubated for 1h prior to ONOO −loading.Propidium iodide (PI)counterstaining indicates nuclear localization (red;λex 543nm,λem 560−615nm).NP3ﬂuorescence was collected at 420−480nm with λex 405nm.least 6h (Figure 5and Figure S20).Moreover,coinciding with the reports that lysosome response is also associated with nitrosative stress,11,52our results demonstrated that enhanced ﬂuorescence is partially located in the lysosomal compartment (Figure S21),suggesting dynamic spatiotemporal coordination between ONOO −and lysosomes.Similar changes in NP3ﬂuorescence were con ﬁrmed in HBMEC after OGD (Figures S22and S23).The results indicated that NP3can monitor ONOO −ﬂux during ischemic injury.Accompanied by increased NP3ﬂuorescence in mitochondria,as well as the spreading distribution in endothelial cells,ONOO −accumu-lation challenged the endothelial cell and contributed to apoptosis,as shown by annexin V staining (blue),in agreement with the results that ONOO −could induce apoptosis (Figure S24).Thus,these collective data demonstrated that NP3e ﬃciently visualized progressive ONOO −ﬂuxes in endothelial cells with excellent temporal and spatial ability.3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bro-mide (MTT)assay showed that NP3did not exhibit cytotoxicity within 48h except at such high concentrations as 150and 200μM (Figure S25).Moreover,the fairly low cytotoxicity of NP3was con ﬁrmed by mitochondrial membrane potential-sensing dye JC-1.Since the loss of mitochondrial transmembrane potential (ΔΨm )signals is a hallmark of mitochondrial dysfunction,cytotoxicity,and apoptotic signal-ing,53we used JC-1to further investigate the potential cytotoxic e ﬀects of NP3on endothelial cells.No signi ﬁcant shift in JC-1ﬂuorescence from red to green was observed following NP3treatment up to 100μM in EA.hy926endothelial cells,compared with control cells undergoing JC-1staining (Figure S26).Thus,these results suggest that NP3is nearly noncytotoxic up to 100μM in endothelial cells,implying excellent biocompatibility for biological application,although further in vivo testing is necessary prior to application for clinical diagnosis.Real-Time Imaging of Endogenous Peroxynitrite Formation after Brain Microvessel Injury in Live bined with in vivo two-photon laser scanning microscopy (TPLSM),NP3enabled visualization of dynamic changes of neurovascular ONOO −formation upon ischemia in live mice.The ischemia mice were modeled by rose bengal-induced vascular occlusion 54or laser irradiation-induced microvessel rupture.55The time series images in Figure 6are individual frames from a continuous time-lapse movie.The dynamic elevation of local ONOO −formation (arrows)in the microvessel indicated by strong NP3ﬂuorescence was e ﬃciently monitored over a recording period of 30s (Figure 6a and Movie S1).In contrast,no signi ﬁcant signal was observed in negative control experiments in which mice were modeled in the same way but not treated with NP3,indicatingFigure 4.Characterization of ONOO −formation by use of NP3in endothelial cells upon OGD.(a,b)Representative confocal images show time-dependent accumulation of NP3ﬂuorescence (green;λex 405nm,λem 420−480nm)in (a)EA.hy926endothelial cells over 0.5−2h following OGD exposure as well as in (b)human brain microvascular endothelial cells (HBMEC).PI counterstaining indicated nuclear localization (red,λex 543nm,λem 560−615nm).(c)The OGD-initiated NP3ﬂuorescence response to ONOO −was modulated by suppressing ONOO −formation.EA.hy926endothelial cells were pretreated with FeTTPS (1μM),W7(1μM),melatonin (10μM),or BAPTA (1μM)1h prior to OGD treatment to suppress the ONOO −signal.the capacity of NP3for in vivo tracking of ONOO −ﬂuxes during brain vascular damage (Figure S27).Moreover,to provide another example of the in vivo-visualized ONOO −ﬂux enabled by NP3,real-time imaging of endogenous ONOO −formation was performed in live mice su ﬀering from laser irradiation-induced cerebrovascular rupture at the two-photon wavelength of 800nm.As shown in Figure 6b,endogenous ONOO −formation increased dramatically upon laser irradiation-mediated microvessel injury at 10s pointand remained at its highest ﬂuorescent intensity for at least 30s (Figure 6b,c and Movie S2).Similar to our previously reported results that ischemia induced ONOO −production based on indirect evidence from immunoblot or immunostaining,10,12we observed laser irradiation-induced local ONOO −formation in injured cerebral microvessels in live mice using the combination of in vivo TPLSM and probe NP3.More importantly,we were able to visualize the dynamic changes of ONOO −formationinFigure 5.Distribution of NP3ﬂuorescence,examined by counterstaining with the mitochondria indicator MitoRed after OGD.(a)Representative confocal images show temporal changes of ONOO −-dependent NP3ﬂuorescence (green;λex 405nm,λem 420−480nm)and MitoRed (red;λex 543nm,λem 560−615nm),as well as the apoptosis maker annexin V (blue;λex 488nm,λem 505−550nm),following OGD treatment.(b)Orthogonal projections onto the x −z (upper)and y −z (right)planes are shown to con ﬁrm the colocalization of NP3and MitoRed throughout endothelial cells as shown in panel a after ischemia injury.All images were captured with a Zeiss LSM 510confocal microscope.a temporal and spatial manner with high-resolution images in live mice.■CONCLUSIONTo conclude,by considering basic principles of probe and drug design,we developed a ﬂuorescent probe with desired pharmacokinetic and photophysical properties suitable for imaging of ONOO −generation in both live cells and live animals.The probe,designed by blocking the ESIPT process of the 2-(2′-hydroxyphenyl)benzothiazole ﬂuorophore with a smart reaction trigger that readily undergoes bioorthogonal and speci ﬁc reaction with ONOO −in biological contexts,features excellent speci ﬁcity,high sensitivity,two-photon excitability,and ready BBB penetrability.Application of this probe to track in situ generation of ONOO −in the brains of live mice has provided direct evidence that vascular occlusion or laser irradiation would induce local ONOO −formation in injured cerebral microvessels.The probe should serve as a powerful imaging tool to explore ONOO −biology under a variety of pathological contexts in vitro and in vivo.Furthermore,electronic and steric e ﬀects on probe sensitivity and the signi ﬁcance of probe pharmacokinetic properties to its real-world application,as considered in this work,should be instructive for future probe design.■ASSOCIATED CONTENT*Supporting Information The Supporting Information is available free of charge on the ACS Publications website at DOI:10.1021/jacs.5b06865.General experimental details for chemistry,photophysical property characterization,and biology materials and methods;probe synthesis and structure characterization (Scheme S1);preparation of various ROS and RNS species;determination of quantum yield and two-photon absorption cross section;photophysical property data (Table S1and Figures S1−S13);supplementary imaging (Figures S14−S27);and 1H NMR,13C NMR,and HRMS spectra of NP2,NP3,and NP4(PDF )Movie 1,showing that dynamic elevation of local ONOO −formation in the microvessel,indicatedbyFigure 6.Real-time visualization of endogenous peroxynitrite ﬂuxes after brain microvessel injury with a combination of NP3and in vivo two-photon laser scanning microscopy.For two-photon imaging,cortical brain vessels 10−15μm in diameter and 100μm below the cortical surface were selected for imaging.(a,b)The time-series images are individual frames from a continuous time-lapse movie and show dynamic NP3ﬂuorescence elevation (arrows)following (a)rose bengal-induced vascular occlusion and (b)laser irradiation-induced vascular rapture in live mice.(c)Mean values of NP3ﬂuorescence intensity from panel b were measured to quantify the progressive ONOO −formation in cerebrovessels after ischemia.Emission was collected at 420−480nm for NP3ﬂuorescence and 575−630nm for dextran Texas or rose bengal upon excitation at 800nm.strong NP3ﬂuorescence,was eﬃciently monitored overa recording period of30s(AVI)Movie2,showing that endogenous ONOO−formationincreased dramatically upon laser irradiation-mediatedmicrovessel injury at10s point and remained at itshighestﬂuorescent intensity for at least30s(AVI)■AUTHOR INFORMATIONCorresponding Authors*changhuahan@(F.H.)*huyz@(Y.-Z.H.)*yhhu@(Y.-H.H.)Author Contributions∥X.L.and R.-R.T contributed equally to this work.NotesThe authors declare no competingﬁnancial interest.■ACKNOWLEDGMENTSThis work was supported in part by Projects of National Natural Science Foundations of 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纳米催化二氧化碳制甲醇英文

纳米催化二氧化碳制甲醇英文Nanocatalysis for the Production of Methanol from Carbon Dioxide.Carbon dioxide (CO2) is a significant greenhouse gas that contributes to global warming. However, converting it into useful chemicals such as methanol offers a sustainable and environmentally friendly approach to mitigate its adverse effects. Nanocatalysis, a field that utilizes nanoscale materials to catalyze chemical reactions, has emerged as a promising technology for this purpose.Nanocatalysis Principles and Applications.Nanocatalysis leverages the unique properties of nanomaterials, including their large surface area and high reactivity, to enhance catalytic activity. These nanomaterials, often in the form of nanoparticles or nanostructures, can significantly improve the rate and selectivity of chemical reactions. In the context of CO2conversion, nanocatalysts can lower the activation energy required for the reaction, making it more energetically favorable.CO2 to Methanol Conversion.The conversion of CO2 into methanol involves a multi-step process known as the methanol synthesis. Typically, this process requires high temperatures and pressures, as well as a suitable catalyst. Nanocatalysts cansignificantly reduce these requirements, making the process more energy-efficient and cost-effective.The most common nanocatalysts used for CO2 hydrogenation to methanol are based on copper. Copper nanoparticles, due to their high activity and selectivity, are particularly effective in promoting this reaction. Other metals, such as palladium and platinum, have also been explored for this purpose.Nanocatalyst Design and Optimization.The design and optimization of nanocatalysts for CO2 conversion are crucial for achieving high catalytic performance. Factors such as particle size, shape, and composition can significantly influence the catalytic activity. For instance, smaller nanoparticles typically exhibit higher catalytic activity due to their increased surface area. Similarly, the choice of support material can also affect the stability and activity of the nanocatalyst.Challenges and Future Prospects.While nanocatalysis offers significant potential for CO2 conversion, several challenges need to be addressed. One of the main challenges is the scalability of nanocatalysts for industrial applications. Current methods for synthesizing nanomaterials are often not suitable for large-scale production. Additionally, the stability of nanocatalysts under reaction conditions is also a concern, as they can often deactivate or agglomerate over time.Future research efforts should focus on developing more stable and scalable nanocatalysts for CO2 conversion.Innovations in nanomaterials synthesis and characterization techniques can help address these challenges. Furthermore, integrating nanocatalysts with other renewable energy sources, such as solar or wind power, can further enhance the sustainability of the process.In conclusion, nanocatalysis holds promise for the efficient conversion of CO2 into methanol. By leveraging the unique properties of nanomaterials, we can develop more effective and sustainable catalysts for this important reaction. Future research in this area could lead to significant advancements in green chemistry and help mitigate the impact of climate change.。

作物种子学专业名词中英文对照

作物种子学专业名词中英文对照中文英文氨基酸Amino acid暗发芽Dark-germination白熟期White ripe stage半活期Half-living period半纤维素Hemicellulose伴胞Companion cell苞片苞叶Bract胞质分裂Cytokinesis Plasmodieresis饱和湿度Saturation humidity保卫细胞Guard cell不发育的胚Rudimentary embryo不健全的种子Unsound seed不良种子Bad seed不实年度Off year不透水性的种子Impermeable seed不育种子Sterile seed infertile seed不正常苗Abnormal seedling层积处理Stratification长寿种子Macrobiotic seeds超低温保存Cryopreservation超干贮藏Ultra-dry storage成苗率Stand establishment percentage成熟Maturation成熟期Maturation phase Maturation period 赤霉素Gibberellin赤霉酸Gibberellic acid GA翅果Samara虫蛀种子Worn-eaten seed出土型发芽Epigeous germination传递细胞Transfer cell传统型种子Orthodox seed雌蕊Pistil次生休眠Secondary dormancy代谢蛋白Metabolic protein单性生殖Parthenogenesis蛋白质体Protein body Aleuroplast蛋白质Protein蛋白质种子Protein seed电导率测定Electrical conductivity test淀粉Starch淀粉粒Starch grain淀粉胚乳Starchy endosperm豆球蛋白Legumin短命种子Microbiotic seeds盾片Scutellum Scutel多核体多核细胞Coenocyte多胚现象Polyembryonia Polyembryony萼片Sepal发芽Germination发芽高峰值Peak value发芽口Micropyle发芽力Germination power Germinating ability 发芽率Germinating percentage发芽势Germinating energy Germinative energy 发芽速率Germinative rate发芽抑制剂Germination inhibitor发芽值Germinative value发芽指数Germinative index非需光的种子Non light-requiring seeds分果Schizocarp粉质种子Starch seed复果聚花果Multiple fruit腹白Abdominal white腹沟Crease高尔基体Dictyosome谷醇溶蛋白Prolamine谷蛋白Glutelin冠毛茸毛Pappus光促进萌发的种子Light promoted seeds光媒介发芽Light-mediated germination光抑制发芽的种子Light-inhibited seeds果胶Pectin果皮Fruit coats Pericarp果脐Fruit navel果实Fruit果糖Fructose合点Chalaza合子Zygote核果Drupe核膜Nuclear membrane Nuclear envelope 核仁Nucleolus Nucleole核糖体Ribosome核型Nuclear type褐熟期Brown ripe stage后熟After-ripening After mature糊粉Aleurone糊粉层Aleurone layer糊粉粒Aleurone grain护颖颖片Glume花Flower花瓣Petal花被Perianth floral envelope 花萼Calyx花粉Pollen花粉管Pollen tube花粉粒Pollen grain花粉囊Pollen sac花冠Corolla花管Floral tube花色素Anthocyanidin花色素苷花青苷Anthocyanin花丝Filament花托Receptacle花药Anther花柱Style黄熟期Yellow ripe stage活力Vigour活力指数Vigour index活种子Viable seed激动素Kinetin极核Polar nucleus忌光性种子Heliophobous seeds加速老化测定Accelerated aging test加速老化法Accelerated aging荚果Legume假发芽False germination假果False fruit假核果False drupe假种皮Aril坚果Nut角质Cutin结构蛋白Structural protein结果率Fruit bearing percentage 结果实Bearing结实年份Crop year bearing year结实周期Periodicity of seed bearing 结种子Seed setting结籽实率Seed setting percentage 聚合果Aggregate fruit绝对湿度Absolute humidity开花花开期Anthesis空粒Blind seed empty seed控制劣变测定Controlled deterioration test枯熟期Dead ripe stage蜡熟期Waxy ripe stage老化Aging老化的种子陈种子Aged seed Old seed离体胚休眠Dormancy of excised embryos劣变Deterioration临界水分Critical water content磷脂Phosphatide绿熟期Green ripe stage麦醇溶蛋白Gliadin麦谷蛋白Glutenin麦芽糖Maltose芒Awn酶Enzyme萌动Protrusion萌发Sprouting萌发迟缓Postponing germination Delayed germination 萌发过程Germination process萌发过程中的种子Germinating seed萌发孔Germination aperture萌发抑制物Germination inhibitor面筋Gluten明线Light line膜的完整性Intactness of membrane膜损伤Membrane damage母细胞Mother cell内稃Palea内果皮Endocarp内胚乳Endosperm内脐合点Chalaza内种皮Endopleura耐藏潜力Storage potential耐藏性Storability能发芽的种子Germinable seed能荷Energy charge EC农业种子Agriculture seeds 胚Embryo 胚柄Suspensor胚根Radicle胚根鞘Coleorhiza胚根原细胞Hypophysis胚囊Embryo sac Embryonic sac胚乳Endosperm胚胎发生Embryogenesis embryogeny胚休眠Dormancy of embryoEmbryo dormancy 胚芽Plumula embryo bud embryonic bud 胚芽鞘Coleoptile胚中轴Embryonic axis胚珠Ovule胚状体Embryoid品种纯度Purity of variety平衡水分Equilibrium moisture content葡萄糖Glucose脐褥Callus funicularis脐条Raphe强迫休眠Enforced dormancy Stressful dormancy 清蛋白白蛋白Albumin球蛋白Globulin球果Cone Strobile全熟的种子Fully ripened seed染色体Chromosome染色体畸变Chromosome aberration人工胚乳Artificial endosperm人工种皮Artificial seed coat人工种子Artificial seed人工贮藏Artificial storage稔实度Seed setting rate日平均发芽量Mean daily germination MDG日平均发芽率Mean day’s germination绒毛Hair乳熟期Milk ripening stage上胚轴Epicotyl上胚轴休眠Epicotyl dormancy上皮细胞Epithelium上位式Epigyny渗透调节Osmotic conditioning生长素Indoleacetic acid生活力Viability生理成熟Physiological ripening生理休眠Physiological dormancy实用发芽率Practical germination percentage 寿命Longevity Life span受精作用Fertilization受伤害的种子Damaged seed瘦果Achene束缚水Bound water衰老Aging Senescence双受精Double fertilization双重休眠Double dormancy水合—脱水Rehydration-dehydration 死种子Dead seed四唑测定Tetrazolium test糖类Carbohydrate体细胞胚Somatic embryo天然贮藏Storage in nature田间出苗Field emergene脱落酸Abscisic acid ABA脱水素Dehydrin外稃Lemma外果皮Exocarp外胚乳Perisperm外胚叶Epiblast外颖Outer glume外源凝集素Lectin外种皮Testa豌豆球蛋白Vicilin丸化Pelleting完全花Complete flower完熟期Full ripe stage顽拗型种子Recalcitrant seed维生素Vitamin未成熟胚Immature embryo未熟粒Immature seed无胚乳种子Exalbuminous seed无胚种子Embryoless seed无氧呼吸Anaerobic respiration吸附性Absorbability吸湿性Hygroscopicity吸胀Imbibition吸胀冷害Imbibition chilling injury吸胀速率Rate of imbibition细胞Cell细胞板Cell plate细胞壁Cell wall细胞分裂素Cytokinin细胞核Cell nucleus细胞间质Intercellular substance细胞膜Cell membrane细胞器Cell Organelle Cell organ 细胞型指胚乳Cellular type细胞质Cytoplasm下胚轴Hypocotyl下胚轴休眠Hypocotyl dormancy下位式Hypogyny纤维素Cellulose酰胺Amide线粒体Mitochondrion相对湿度Relative humidity小坚果Nutlet小穗Spikelet小穗轴Rachilla Rhachilla雄蕊Stamen pl. Stamina休眠期Dormancy stage Dormant period 需光种子Light-requiring seeds延迟发芽的种子Sluggish seed delayed seed衍生细胞Derivative cell胰蛋白酶抑制剂Trypsin inhibitor乙烯Ethylene颖果Caryopsis Grain硬实种子Hard seed油脂酸败Rancidity油质种子Oil seed有胚乳种子Albuminous seed有色体Chromoplast有丝分裂Karyokinesis有氧呼吸Aerobic respiration幼苗活力分级法Seedling vigor classification 幼苗评价Seedling evaluation诱导休眠Induced dormancy原初休眠Primary dormancy原胚Proembryo Primary embryo 原始生活力Primary viability原始细胞Initial cell原种Basic seed沼生目型指胚乳Helobial type蔗糖Sucrose真空贮藏Vacuum storage整齐度、均匀性Uniformity正常苗Normal seedling支链淀粉Starch branching脂肪Oil脂肪酸Fatty acid脂肪体Lipid body直链淀粉Amylose植物激素Phytohormone Plant hormone 纸间发芽法Between paper BP纸上发芽法Top of paper TP中果皮Mesocarp中间细胞Intermediate cell中间型种子Middle seed中胚轴Mesocotyl中寿种子Mesobiotic seeds种被休眠Coat imposed dormancy种柄Seed stalk Seed-pedicels种翅Seed wing种阜Caruncle Strophiole种苗活力Seed and seedling vigour种苗评价测定法Seedling evaluation test种苗生长测定Seedling growth test种胚Embryo种皮Seed coat种脐Hilum Pl. hila种衣剂Seed coating formulation种质库Germplasm bank种质资源Germplasm resources种子Seed种子包衣Seed coating种子包装Seed package种子保存Seed preservation种子标准化Seed standardization种子处理Seed treatment种子带Seed tapes种子的结露Seed dewfall种子发芽Germination of seed种子发芽力Seed germinability种子发芽潜力Seed germination potential种子干燥Seed drying种子工程Seed engineering种子呼吸Seed respiration种子活力Seed vigour种子精选Seed choice种子生活力Seed viability Viability of seeds 种子生理Physiology of seeds种子生理学Seed Physiology种子生命力Seed Vitality Vitality of Seeds 种子生物学Seed Biology种子毯Seed mats种子丸Seed pellets种子微生物Microorganism of seed种子休眠Seed dormancy种子学Seed Science种子真实性Genuineness of seed种子质量Seed quality种子贮藏Seed storage皱粒Shriveled seed Wrinkle seed 珠被Integument Integumentum 珠孔Micropyle珠心Nucellus Nucella主要贮藏蛋白质Major storage protein柱头Stigma子房Ovary子叶Cotyledon自由水Free water最终发芽率Final germination percentage。

黑果枸杞花色苷的提取、纯化及降解动力学研究

连敏，高艺玮，年新，等. 黑果枸杞花色苷的提取、纯化及降解动力学研究[J]. 食品工业科技，2024，45（6）：24−31. doi:10.13386/j.issn1002-0306.2023080105LIAN Min, GAO Yiwei, NIAN Xin, et al. Study on Extraction, Purification and Degradation Kinetics of Anthocyanins from Lycium ruthenicum [J]. Science and Technology of Food Industry, 2024, 45(6): 24−31. (in Chinese with English abstract). doi:10.13386/j.issn1002-0306.2023080105· 特邀主编专栏—枸杞、红枣、沙棘等食药同源健康食品研究与开发（客座主编：方海田、田金虎、龚桂萍） ·黑果枸杞花色苷的提取、纯化及降解动力学研究连　敏，高艺玮，年　新，王梦泽*（宁夏大学食品科学与工程学院，宁夏银川 750021）摘　要：以花色苷提取量为主要考察指标，通过单因素和正交试验优化冻干黑果枸杞花色苷提取工艺，并在此条件下研究花色苷纯化工艺及其降解动力学，探讨不同温度、pH 下花色苷提取量的变化。

结果表明，提取最佳工艺条件为：料液比1:25（g :mL ）、乙醇浓度60%、pH4、提取时间2 h ，此条件下花色苷提取量达36.507±0.325 mg/g 。

研究显示AB-8大孔树脂纯化黑果枸杞花色苷效果最好，对花色苷吸附量和解吸量的影响效果最佳，其最佳条件为：上样液浓度200 mg/100 g ，解吸乙醇浓度80%，上样流速2 mL/min ，洗脱流速2 mL/min ，上样体积为5 BV ，纯化率为90.02%。

Process for the production of anthocyanin

专利名称：Process for the production of anthocyanin 发明人：平尾壽啓,小林昭雄,岡澤敦司,原田和生,平田收正申请号：JP2011081010申请日：20110331公开号：JP5869773B2公开日：20160224专利内容由知识产权出版社提供摘要：PROBLEM TO BE SOLVED: To provide a production method of anthocyanin which safely and easily increase the anthocyanin in a plant body.SOLUTION: There is provided the production method of the anthocyanin which is characterized in that an anthocyanin-producing plant is an exposed to green leaf volatile component, and the anthocyanin-producing plant is exposed to methyl jasmonate. There is also provided the anthocyanin-producing plant. In the production method anthocyanin, the green leaf volatile component is one of n-hexanol, Z-3-hexenol, Z-3-hexenal, E-2-hexenol, n-hexyl acetate, Z-3-hexenyl acetate, n-hexanal, E-2-hexanal, n-nonanol and n-nonanal.申请人：株式会社フジキン,国立大学法人大阪大学地址：大阪府大阪市西区立売堀２丁目３番２号,大阪府吹田市山田丘１番１号国籍：JP,JP代理人：清原義博更多信息请下载全文后查看。

青霉素发明者英语作文

青霉素发明者英语作文In the annals of medical history, the discovery of penicillin stands as a monumental breakthrough that has saved countless lives. This essay delves into the life of its inventor, Sir Alexander Fleming, and the serendipitous discovery that changed the course of modern medicine.Sir Alexander Fleming was a Scottish bacteriologist born in 1881. His interest in medicine was piqued at an early age, leading him to pursue a career in the field. After serving in World War I as a captain in the Royal Army Medical Corps, Fleming returned to civilian life and continued his work in research.The discovery of penicillin was accidental, occurring in 1928. Fleming had left a petri dish of Staphylococcus bacteria uncovered in his laboratory at St. Mary's Hospital in London. Upon returning from a two-week vacation, he noticed that a mold had contaminated the dish. What was remarkable was that the bacteria surrounding the mold had been killed, while the bacteria farther away were alive. This mold was thePenicillium notatum, and it produced a substance that provedto be the first true antibiotic.Fleming's discovery was groundbreaking, but it was not immediately recognized for its full potential. It was notuntil the late 1930s that a team of scientists, including Howard Florey and Ernst Boris Chain, began to explore thepractical applications of penicillin. Their work led to the large-scale production of the antibiotic, which was crucial during World War II when it was used to treat woundedsoldiers and prevent infections.The impact of penicillin on medicine cannot be overstated. It has been used to treat a wide range of bacterial infections, from strep throat to pneumonia. The discovery has also paved the way for the development of other antibiotics, which have become essential tools in the fight against bacterial diseases.In conclusion, Sir Alexander Fleming's accidental discovery of penicillin was a turning point in the history of medicine. His work has had a profound impact on public health and continues to influence the development of new treatments for bacterial infections. The story of penicillin serves as a reminder of the importance of curiosity and the potential for unexpected discoveries to change the world.。

杂交构树生产性能、营养价值及饲喂效果分析研究

16 ·2023.70 引言杂交构树，由中国科学院植物研究所利用现代生物技术和传统育种方法培育[1]，具有优质蛋白含量高，氨基酸、维生素、微量元素可与苜蓿媲美的营养特点[2]；收稿日期：2023-05-24基金项目：云南省重大科技专项（202002AE320011）；大理州创新引导与创新能力提升（D2021ZX01）作者简介：华世旋（1996-），男，本科，助理畜牧师，从事饲草饲料开发研究与推广应用工作。

*通信作者简介：杨旭艳（1987-），女，硕士，畜牧师，从事畜牧生产发展科技推广应用工作。

华世旋，杨旭艳，董红林，等．杂交构树生产性能、营养价值及饲喂效果分析研究[J]．现代畜牧科技，2023，98（7）：16-20．　doi ：10. 19369/j. cnki.2095-9737. 2023. 07. 004．　HUA Shixuan ，YANG Xuyan ，DONG Honglin ，et al ．Analysis and Study on the Production Performance ，Nutritional Value and Feeding Eﬀect of Broussonetia Papyrifera [J]．Modern Animal Husbandry Science & Technology ，2023，98（7）：16-20．杂交构树生产性能、营养价值及饲喂效果分析研究华世旋1，杨旭艳1*，董红林1，熊跃1，施雪明1，陈艳萍2（1. 云南省大理白族自治州畜牧工作站，云南大理 671000；2. 云南省祥云县畜牧工作站，云南大理 672100）摘要：本研究共设定4个试验内容，从多个方面来分析杂交构树用作饲草饲料的价值与效能。

杂交构树生产性能测定试验设置杂交构树、王草、青贮玉米—饲用燕麦轮作3个处理，测定鲜草产量及化学营养品质，对比分析杂交构树生产性能，分析各生产指标与杂交构树添加比例的关系。

江苏省无锡市天一中学2024年高三英语第一学期期末检测模拟试题含解析

江苏省无锡市天一中学2024年高三英语第一学期期末检测模拟试题注意事项：1．答卷前，考生务必将自己的姓名、准考证号填写在答题卡上。

2．回答选择题时，选出每小题答案后，用铅笔把答题卡上对应题目的答案标号涂黑，如需改动，用橡皮擦干净后，再选涂其它答案标号。

回答非选择题时，将答案写在答题卡上，写在本试卷上无效。

3．考试结束后，将本试卷和答题卡一并交回。

第一部分（共20小题，每小题1.5分，满分30分）1．With Mother's Day around the corner，I have taken some money out of thebank_____________ presents for my mother.A．buy B．to buyC．buying D．having bought2．People all think it strange that the boy should tell what’s written on the paper in another room without looking at it. It really ________ explanation.A．prevents B．challenges C．interrupts D．confuses3．—Why didn’t you try to finish the composition?— I tried to, but ________ I could, the bell ran announcing the end of the examination. A．until B．whenC．after D．before4．The purpose of her talking to me last night actually _______this: That I shall never trust him any more in future.A．comes round B．comes out C．comes on D．comes to5．_____ is often the case, we have worked out the production plan.A．Which B．When C．What D．As6．________ by many potential customers, the salesman had to gather his courage and sell the product in different ways.A. Having denied B．DenyingC．Being denied D．Having been denied7．--- Is it convenient to you if I call you up at 9 o’clock tomorrow morning?--- I’m afraid no t. I ______ a meeting then.A．will have attended B．was attendingC．will be attending D．am attending8．----What happened to the young trees we planted last week？---The trees_____________ well，but I didn't water them.A．might grow B．needn't have grownC．would grow D．would have grown9．— Hello, I ________ to ask if I can book a flight ticket to Hainan tomorrow? —Sorry, we’ve already sold out.A．phone B．will phoneC．am phoning D．have phoned10．Jack and Mike were found cheating in the exam, and_______by their teacher at the moment.A．were scolded B．are being scoldedC．have been scolded D．were being scolded11．The maple trees turn a brilliant red in autumn, adding another to the colors in the harvest season.A．theme B．versionC．category D．dimension12．It is through years of research ________ scientists have discovered the relationship between social media addiction and depression.A．since B．before C．that D．when13．—I was informed that you won the lottery?—Are you kidding? Maybe I will win a lottery when______.A．you cry for the moon B．pigs flyC．all good things come to an end D．you get a new lease on life14．Some schools, including ours, will have to make ________ in agreement with the national soccer reform.A．amusements B．adjustmentsC．appointments D．achievements15．--- How did you like the concert last night?--- I enjoyed it very much, but the dancers ______ a better job.A．could have done B．couldn’t have done C．could do D．had done16．As a doctor, I spend most of the time with my patients, and t hat’s ________ it is in my day.A．how B．when C．why D．where17．—What’s up? Y ou look worried.—Well, I ______ on the problem for 5 hours but I haven’t got a single clue.A．have worked B．workedC．will work D．have been working18．Citizens are _____ to exercise their rights, but under no circumstances can they violate other people’s rights.A．on track B．on scheduleC．at ease D．at liberty19．What a pity! ________joined in the party, I would have met my old friend．A．lf I have B．Had I C．I had D．Have I20．– She will finish the project within three days!-- ______________! I could do it in three hours.A．You are right. B．I’m stuck. C．Oh, come on D．Don’t mention it.第二部分阅读理解（满分40分）阅读下列短文，从每题所给的A、B、C、D四个选项中，选出最佳选项。

2024-2025学年重庆市SAGSSAT普通高等学校招生高三上学期7月适应性考试英语试题

2024-2025学年重庆市SAGSSAT普通高等学校招生高三上学期7月适应性考试英语试题Want to know all the ins-and-outs regarding the Paris 2024 Olympics? Then you’ve come to the right place! Below you will find all the information about the new Olympic sports.Olympic skateboardingWho will follow in the footsteps of Momiji Nishiya, the first Olympic skateboarding champion, who was crowned at the last Games at the age of 13? The answer will be revealed in Paris this summer. Speed, technique, mastery of the board will be required to successfully perform the most beautiful tricks.Olympic sports climbingWith its second time entering the Olympics, sports climbing has three branches: Speed, Bouldering (抱石攀岩) and Lead. Speed is all about the speed of climbing. Bouldering is all about climbing a boulder on a wall and Lead requires athletes to climb a difficult route 20 meters high.Olympic surfingThe Olympics will be held in Paris, but what about Olympic surfing? There is one venue (场地) that is literally on the other side of the world. The location Teahupoo, on the island of Tahiti, is where Olympic surfing will take place. This island is part of French Polynesia, between Australia and South America.Olympic breakdancingBreakdancing is the new Olympic sport that will make its first appearance in Paris 2024. How to establish a grading system without taking away the artistic freedom at the same time? The points should be based on creativity and musicality, a gray area that depends a lot on feeling and thus has a lot of subjectivity to it.Looking for a handy list of all sports? Click here! Throughout Paris there are all kinds of stadiums and arenas (竞技场) where sporting events are held. Not only Paris itself, but also the surrounding suburbs of the capital of France set the stage for the Olympic Games!1. Which sport is entering the Olympics for the first time in Paris 2024?A．Olympic skateboarding. B．Olympic sports climbing.C．Olympic surfing. D．Olympic breakdancing.2. Which of the following statements is correct?A．Olympic skateboarding requires a high level of physical strength.B．Three branches of Olympic sports climbing evaluate various sides of skills.C．All the above four Olympic events will be held in Paris.D．The scoring of Olympic breakdancing is highly objective.3. Where is the text probably taken from?A．A sports website. B．A health magazine.C．A travel brochure. D．An Olympic news report.A letter written by Charles Darwin in 1875 has been returned to the Smithsonian Institution Archives（档案馆） by the FBI after being stolen twice."We realized in the mid-1970s that it was missing," says Effie Kapsalis, head of the SmithsonianInstitution Archives. "It was noted as missing and likely taken by an intern（实习生）， from what the FBI is telling us. Word got out that it was missing when someone asked to see the letter for research purposes," and the intern put the letter back. "The intern likely took the letter again once nobody was watching it."Decades passed. Finally, the FBI received a tip that the stolen document was located very close to Washington, D. C. Their art crime team recovered the letter but were unable to press charges because the time of limitations had ended. The FBI worked closely with the Archives to determine that the letter was both authentic and definitely Smithsonian's property.The letter was written by Darwin to thank an American geologist, Dr. Ferdinand Vandeveer Hayden, for sending him copies of his research into the geology of the region that would become Yellowstone National Park.The letter is in fairly good condition, in spite of being out of the care of trained museum staff for so long. "It was luckily in good shape," says Kapsalis, "and we just have to do some minor things in order to be able tounfold it. It has some glue on it that has colored it slightly, but nothing that will prevent us from using it. After it is repaired, we will take digital photos of it and that will be available online. One of our goals is to get items of high research value or interest to the public online."It would now be difficult for an intern, visitor or a thief to steal a document like this. "Archiving practices have changed greatly since the 1970s," says Kapsalis," and we keep our high value documents in a safe that I don't even have access to."4. When did the Smithsonian Institution Archives realize that this letter was lost ?A．In 1875. B．In 1885.C．In the mid-1970s. D．In the late 1970s5. Who ultimately owns this letter ?A．FBI. B．Smithsonian Institution Archives.C．Charles Dsrwin. D．Dr. Ferdinand Vandeveer Hayden.6. What is the purpose of writing this letter ?A．In commemoration of his discovery of Yellowstone National Park.B．To thank himself for publishing a regional geological research.C．In order to obtain copies of his research.D．To express gratitude to the geologist.7. What will Smithsonian Institution Archives do according to Kapsalis?A．Stain the letter. B．Train museum staff.C．Keep it a permanent secret. D．Make it available online.Anime（二次元） culture has become a very common culture in our daily life. As people's minds become more and more open, most people gradually begin to accept anime culture. Although increasingly more young people like anime culture than before, many still don't know why there are so many people like anime culture in reality. What is its attractive charm?First and foremost, anime is not immature and cynical（玩物丧志的）. Many people don't understand people who like anime, just because they don't try to understand anime. They only evaluate it through other people's words and their own knowledge. Of course, the comments are not very pleasant. After all, the older generation don't agree with anime.Moreover, the reason why today's young people like anime so much is not because they are killing time, nor because they are addicted to animation, but because of pressure. Today's society is completely different from that of the 1980s. Back then, having a motorcycle or a big TV at home was already impressive. But now, cars, houses, and savings are everywhere, and young people are easily suffocated（扼制）.When we see anime protagonists invincible in the drama, willing to sacrifice everything to defeat enemies for their dreams and friends, it's like seeing us unwilling to be outdone in reality. Aren't those bosses in anime just like the things that bring us pressure in reality? When we see anime protagonists hitting them one after another, it's like we're putting all the pressure on ourselves, and we feel exhilarated（畅快淋漓）.In recent years, the rapidly rise popularity of anime games such as Genshin Impact（原神） and Love of Light and Night（光与夜之恋） has certainly brought a rich side to the life of the current young generation. Luo Xiang once said: "Paper people are virtual, and after a long time in the virtual story, they are unwilling to enter the real story." It is normal to embrace anime cultural , but we still need to have expectations for the future and believe in our youth and dreams.8. What is the function of the first paragraph ?A．To enhance people's awareness of anime culture.B．To persuade people stop contacting anime culture.C．To lead the deeper description of the anime trend.D．To arise reader's love to anime culture.9. What is the seniors' view of anime?A．Unbearable. B．Negative.C．awful. D．wonderful.10. What are the reasons why young people like anime drama ?A．Young people cannot bear the enormous pressure of house loans.B．Young people are often oppressed by their parents and elders.C．Young people have lost confidence and hope in the current reality of life.D．Young people have found their dream selves through the characters.11. What can we infer from what Luo Xiang put in the article ?A．We cannot treat the virtual world as an Eden to escape real-life problems.B．The current world is too false and not worth our time to pay attention to.C．In order to let people return to reality, all anime games should be banned.D．The anime world is as important as the real world.Recently, Ai Peiyan, Wei Guo and other scientific researchers from the Medicinal Plant Germplasm Innovation and Utilization Team of the Agricultural and Biotechnological Center of South China Botanical Garden, Chinese Academy of Sciences（中国科学院）， used the "cell factory" method to produce anthocyanins（花青素）， and the output reached 96.23 mg/g(dry weight).In traditional methods, anthocyanins are mainly extracted from plants, while the content of anthocyanins in common fruit fruits is 0.08-25.77 mg/g(dry weight). In addition to low content and complicated extraction process, there are also difficulties such as long plant growth cycle. If a fully synthetic approach is adopted, there are issues such as high cost and difficulty. The team utilized the regulatory mechanism of efficient synthesis and accumulation of anthocyanins in black fruit goji berries, and employed metabolic engineering and other methods to efficiently synthesize high-value anthocyanins in a "cell factory".The team achieved a breakthrough in the efficient synthesis of fruit anthocyanin main component petanin from suspension callus tissue through multi omics（组） joint analysis, based on the established efficient genetic transformation system of black fruit goji berries in the early stage, and achieved a high yield of 96.23 mg/g (dry weight) of anthocyanin through a series of measures.This technology system breaks through the temporal and spatial limitations of the growth cycle and planting area of black fruit goji berries, achieving efficient synthesis under the conditions of a "cell factory", and providing technical support for the protection of wild germplasm resources of black fruit goji berries and the improvement and efficiency of the anthocyanin industry.In addition, the team closely focused on the research and development goals of functional gene mining and industrial development and utilization of anthocyanins in black fruit goji berries. They identified multiple key function al genes that regulate anthocyanin synthesis in black fruit goji berries and established a technical system for efficient production of anthocyanins in black fruit goji berries using suspended callus tissue, laying a solid technical foundation for the development and utilization of anthocyanins in food, health products, and other fields.12. What is the biggest advantage of using a "cell factory" method to produce anthocyanins ?A．The lowest price.B．The steep increase in yield.C．The biggest financial suppose from the local government.D．The simpler collection steps.13. What does the underlined word in the second paragraph mean?A．compose. B．experiment. C．facility. D．method.14. Which of the following statements is correct ?A．Genetic transformation is a must-use technology to reach the success.B．The research team used brown fruit goji berries to produce anthocyanins.C．The plants that used in the research can grow anywhere on Earth.D．The application value of this technology in the food industry is negligible.15. Which can be the best title of this passage ?A．Future humans will achieve the freedom of anthocyanins.B．Anthocyanins- A New Breakthrough in Plant Science.C．Black fruit goji berries give wings to botanic scientific development.D．Scientists Achieve Efficient Production of Anthocyanins Using "Cell Factory"Does your teenager seem addicted to the Internet? Probably yes. 16 At first, parents welcome the Internet into their homes, believing they are opening up an exciting new world of educational chances for their children. 17 Instead of using the Internet for homework or research, they are spending hours messaging with friends, playing online games or talking to strangers in chat rooms. Keeping a healthy balance between entertainment media and other activities in their children’s lives has always been a challenge for parents. The Internet has made this challenge even more difficult.18However, parents are usually not aware that there is a problem until it becomes serious. This is because it is easy to hide what you are doing online and Internet addiction is not widely recognized by the medical community.Children and young people can easily become hooked on online activities such as multi-player games, instant messaging and chat rooms. The most vulnerable children are those who are unpopular or shy with peers. 19 Boys, in particular, are frequent users of online role-playing games, where they assume new identities and interact with other players. For the children who are not so outgoing, playing these games with thousands of other users may apparently be a social activity. 20 . So parents need to help children limit the online time to an appropriate amount.The Unlikely FriendshipIn the heart of the city stood an old library, a place where time seemed to slow down. Among its many visitors was a young girl named Lily, who ________ spent her afternoons there, lost in the pages of books. One day, she noticed a man sitting quietly in a corner, surrounded by stacks of books. His name was Mr. Thompson, a retired professor who had a passion for literature and history.Lily was initially ________ by the man's presence, but soon she found herself draw n to the same corner every day. Mr. Thompson, noticing her curiosity, began to share stories from the books he was reading. Lily listened intently, fascinated by the tales of ancient civilizations and heroic deeds. Their conversations became a ________ part of her visits, and before long, a friendship began to________. Despite their age difference, they discovered that they shared a love for knowledge and a desire to understand the world. Mr. Thompson taught Lily about the importance of ________ and how it could open doors to new ________.As weeks turned into months, Lily's visits to the library became less frequent due to school comments. Feeling the absence of their friend, Mr. Thompson decided to write her letters, sharing interesting facts and insights he came across in his readings. These letters became a source of________ for Lily during stressful times, reminding her of the joy that could be found in the pursuit of knowledge. When summer arrived, Lily returned to the library more ________. She was surprised to find Mr. Thompson waiting for her with a special gift—a first edition of one of her favorite books. Touched by the gesture, Lily realized that their friendship had grown ________ the walls of the library. She decided to ________ Mr. Thompson to her family, introducing him to a world outside of books that was filled with laughter and warmth. Mr. Thompson, in turn, was delighted to be welcomed into Lily's family. He began to accompany them on outings, sharing his vast knowledge and enriching their experiences with historical context and cultural insights. Their bond deepened, and they both found that ________ wasn't limited to the pages of books but could be found in the connections we make with others.As years passed, Lily grew older and ________ to pursue her dreams. But she never forgot the lessons she learned from her unlikely friend. Whenever she felt lost or uncertain, she would remember Mr. Thompson's words of wisdom and find the strength to ________. And whenever she ________ her hometown, she would make sure to stop by the library, hoping to find Mr. Thompsonsitting quietly in his corner, ready to share another story. Their friendship had ________ a lifelong lesson: that the most meaningful connections can come from the most unexpected places.21.A．frequently B．rarely C．occasionally D．never22.A．intrigued B．frightened C．annoyed D．disappointed 23.A．minor B．major C．regular D．irregular24.A．decline B．flourish C．disappear D．wane25.A．competition B．education C．entertainment D．exploration 26.A．experiences B．jokes C．boys D．mistakes27.A．comfort B．confusion C．concern D．conflict28.A．over B．up C．beyond D．on29.A．introduce B．challenge C．recommend D．present30.A．rapid B．fast C．gradually D．regularly31.A．adventure B．friendship C．hardship D．solitude32.A．ran away B．moved away C．passed away D．went away 33.A．give up B．stand still C．move forward D．look back 34.A．encountered B．visited C．left D．found35.A．taught B．ignored C．questioned D．rejected阅读下面短文，在空白处填入1个适当的单词或括号内单词的正确形式。

马铃薯类黄酮_3_O_葡萄糖基化酶基因的克隆...

收稿日期:2009-05-20基金项目:国家自然科学基金资助项目(30860033);江西省教育厅科技项目(2007-313)作者简介:卢其能(1968-),男,江西宜春人,博士,副教授,主要从事植物色素分子遗传与基因工程方面的研究。

马铃薯类黄酮232O 2葡萄糖基化酶基因的克隆与表达分析卢其能1,2,杨　清2,沈春修1(1.宜春学院生命科学与资源环境学院,江西宜春　336000;2.南京农业大学生命科学学院生化与分子生物学系,江苏南京　210095) 摘要:根据茄科植物花色苷生物合成相关基因的资料,设计简并引物,通过RT 2PCR 的方法从马铃薯(Solanumtuberosum cv.Chieftain )紫色芽的cDNA 中克隆到类黄酮232O 2葡萄糖基化酶基因(3GT )的全长cDNA 。

序列分析表明,这个3GT 基因编码的多肽为448个氨基酸残基,在氨基酸水平上与茄科的矮牵牛和茄子的一致性最高,均为76%,多重比较和系统发育分析均表明,该基因为3G T 家族中的一个新成员。

用半定量RT 2PCR 进行了表达分析表明,3GT 在根、叶和块茎中的表达量远高于茎和花;此外,3GT 在Chieftain 的红色愈伤组织中表达而在非红色愈伤组织中不表达,可见,3GT 的表达与花色苷的积累是相关的。

关键词:花色苷;cDNA 克隆;表达模式;Solanum tuberosum ;类黄酮232O 2葡萄糖基化酶中图分类号:Q785 文献标识码:A 文章编号:1000-7091(2009)04-0011-06Cloning and Expression Analysis of Flavonoid32O 2gulcosyltransferase G ene from PotatoLU Qi 2neng 1,2,Y ANG Qing 2,SHE N Chun 2xiu 1(1.C ollege of Life Sciences ,Res ources and Environment Sciences ,Y ichun University ,Y ichun 336000,China ;2.Department of Biochemistry and M olecular Biology ,C ollege of Life Sciences ,Nanjing Agricultural University ,Nanjing 210095,China )Abstract :The degenerate primers were designed based on the published information of sequence of S olanaceaeplants.The cDNA clones encoding UDP 2glucose :flav onoid 32O 2glucosyltrans ferase (3GT )have been is olated fromSolanum tuberosum cv.Chieftain with the degenerate primers by RT 2PCR.The cDNA clone of 3GT encodes a predictedpolypeptides of 448amino acids ,and the deduce protein showed 76%highest identity with 3GT of Petunia hybrida andSolanum melongena ,and the multiple alignment and phylogenetic analysis dem onstrated that it belong to a new member ofthe corresponding 3G T 2family enzymes.The mRNA expression analysis dem onstrated that the accumulation of 3GT tran 2scripts in root ,leaf and tuber was much m ore than that in stem and flower.The accumulation of 3GT transcripts in red 2pigmented callus was much m ore than that in non 2pigmented callus of S.tuberosum cv.Chieftain.The expression of 3GT gene followed by anthocyanin accumulation.K ey w ords :Anthocyanins ;cDNA cloning ;Expression pattern ;Solanum tuberosum ;UDP 2glucose :flav onoid 32O 2glu 2cosyltrans ferase (3G T ) 花色苷是植物次生代谢过程中产生的类黄酮物质,它是花色素与糖以糖苷键结合而成的一类化合物,广泛存在于植物各种组织和器官细胞的液泡中,使其呈现从红、紫到蓝等的不同颜色[1]。

进步的完成设计以提高Bhagyam PCP运行寿命说明书

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