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Mapping of quantitative trait loci corroborates independent genetic control of apple size and shape

Scientia Horticulturae 174(2014)126–132Contents lists available at ScienceDirectScientiaHorticulturaej o u r n a l h o m e p a g e :w w w.e l s e v i e r.c o m /l o c a t e /s c i h o r tiMapping of quantitative trait loci corroborates independent genetic control of apple size and shapeYuansheng Chang a ,Rui Sun a ,Huanhuan Sun a ,Yongbo Zhao b ,Yuepeng Han c ,Dongmei Chen b ,Yi Wang a ,Xinzhong Zhang a ,∗,Zhenhai Han a ,∗aInstitute for Horticultural Plants,College of Agronomy and Biotechnology,China Agricultural University,Beijing 100193,China bChangli Institute for Pomology,Hebei Academy of Agricultural and Forestry Science,Changli,Heibei 066600,China cWuhan Botanical Garden,The Chinese Academy of Sciences,Wuhan 430074,Chinaa r t i c l ei n f oArticle history:Received 3April 2014Received in revised form 18May 2014Accepted 19May 2014Available online 9June 2014Keywords:Fruit shape Fruit size QTLMalus domesticaa b s t r a c tFruit size and shape are important external quality traits in commercial crops.To determine the genetic relationship between the size and shape of apple fruits,quantitative trait loci (QTLs)for apple size (average weight),length,diameter,and shape (length/diameter ratio)were identiﬁed and mapped in progeny of a ‘Jonathan’×‘Golden Delicious’cross.Fruit size,length,and diameter followed a normal distribution.There was no correlation between apple size and shape,but both variables were signiﬁcantly correlated with length and diameter.Forty-ﬁve QTLs for apple size,length,diameter,and shape were mapped to 13chromosomes of the two parent cultivars.Of these,12QTLs for fruit length and diameter either overlapped or were closely associated with QTLs for fruit size,whereas three co-localized with QTLs for fruit shape.No QTLs for fruit size mapped to the same or neighboring regions as QTLs for fruit shape,suggesting that size and shape are under independent genetic control.©2014Elsevier B.V.All rights reserved.1.IntroductionFruit size and shape are important external quality traits in commercial fruit crops.Fruit size is usually quantiﬁed by average weight and determined by fruit length and diameter.Fruit shape can be quantiﬁed morphometrically by length and diameter or can be described using morphological attributes,such as the fruit shape index (FSI;length:diameter ratio),indentation area,and boundary angles (Brewer et al.,2006;Gonzalo et al.,2009).New apple (Malus domestica )varieties,with improved and novel quality traits,for use in apple breeding programs should satisfy consumers (Meneses and Orellana,2013).The market usually demands large fruit.Con-sumers prefer fruit with a relatively larger longitudinal length and smaller latitudinal diameter (Tabatabaeefar et al.,2000;Waseem et al.,2002;Sadrnia et al.,2007).Apple size and shape are under polygene control and are quan-titatively inherited (Brown,1960).The heritability for apple fruit shape aspect (ratio of height to maximum width)is estimated to be 0.79;fruit aspect is best predicted by the ratio of length to∗Corresponding authors.Tel.:+861062734391;fax:+861062734391.E-mail addresses:zhangxinzhong999@ (X.Zhang),rschan@ (Z.Han).diameter (R 2=0.97)(Currie et al.,2000).In our previous work,we identiﬁed ﬁve major genes involved in the segregation of FSI,and the heritability of these genes was as high as 75.0%(Sun et al.,2012).The heritability of length and diameter in strawberry (Fragaria ×ananassa Duch.)is reported as 0.51and 0.21,respec-tively (Lerceteau-Köhler et al.,2012).In hybrid crosses of European pears,the heritability of fruit shape is estimated to be 0.66from parent–offspring regression,and 0.68from variance component analysis (White et al.,2000).The heritability of apple size has been estimated to be as low as 0.33,whereas estimates for fruit weight are higher (0.56–0.61)(Durel et al.,1998;Oraguzie et al.,2001;Alspach and Oraguzie,2002).Phytohormones and environmental factors have different effects on apple fruit length and diameter.Young seeds likely provide a source of gibberellins during the early stages of fruit development (Garcia-Martinez et al.,1987).Application of exoge-nous gibberellic acid (GA 4+7)during blooming or early fruit developmental stages produces longer apples at ripening,with a FSI >1.0in the ‘Golden Delicious’variety (Eccher and Boffelli,1981).In contrast,foliar application of the plant growth retardant paclobu-trazol (PP333)at 1500or 3000ppm,administered 21days after full blooming,resulted in a signiﬁcantly lower FSI in ripe fruit compared with control fruit;the reduced FSI persisted until the fourth year after spraying (Greene,1986).Foliar application of GA 3or GA 4+7/10.1016/j.scienta.2014.05.0190304-4238/©2014Elsevier B.V.All rights reserved.Y.Chang et al./Scientia Horticulturae174(2014)126–132127counteracted the effect of PP333(Curry and Williams,1983).Con-tinuous fruit growth,from cell division to ripening,is primarily associated with auxin-related cell expansion(Devoghalaere et al., 2012).The harvest weight of apples is closely correlated with seed number(including aborted seeds),and increased fruit weight is attributed to increased cell number rather than cell size(Denne, 1963).Environmental factors can also affect apple fruit shape, speciﬁcally temperature and humidity.Shaw(1914)observed that fruit length was longer when temperatures were lower following full bloom.Tromp(1990)reported that the FSI of‘Golden Delicious’was lower in apples grown at a relative humidity between40and 50%than in apples grown at80–90%relative humidity.Developmental rhythms differ for fruit length vs.diameter.The expression of genes important in cell division(e.g.,MdANT1and MdANT2)is high from bloom until15days after full blooming(Dash and Malladi,2012),a period coinciding with active cell division and rapid longitudinal fruit growth(Skene,1966).Quantitative trait loci(QTLs)are important in the investiga-tion of the genetic control of economically valuable traits.Genetic linkage maps enable the identiﬁcation of chromosome regions con-taining one or more genes associated with QTLs(Meneses and Orellana,2013;Tanksley,1993).Since the generation of theﬁrst integrated apple linkage map (Rome Beauty×White Angel;Hemmat et al.,1994),several genetic linkage maps have been reported in apple(Conner et al.,1997; Maliepaard et al.,1998;Liebhard et al.,2002,2003;Baldi et al., 2004;Silfverberg-Dilworth et al.,2006;Calenge et al.,2004;Kenis et al.,2008;Zhang et al.,2012).Saturated and high-density genetic linkage maps are useful for genetic research,and many traits have been mapped in apple (Conner et al.,1997;Weeden et al.,1994;Stankiewicz-Kosyl et al., 2005;Fernández-Fernández et al.,2008;Gao et al.,2005).In apple,QTLs for fruit length have been mapped on linkage group(LGs)2,6,15,and17;and QTLs for apple diameter on LGs2, 5,9,10,and17(Kenis et al.,2008).However,mapping results in dif-ferent years(2004and2005)were found to be inconsistent(Kenis et al.,2008).Several QTLs for apple fruit size have been identiﬁed in different mapping populations,including‘Fiesta’×‘Discovery,’‘Telamon’×‘Braeburn’,‘Royal Gala’×‘Braeburn,’and‘Starkrim-son’×‘Granny Smith’(Liebhard et al.,2003;Kenis et al.,2008; Devoghalaere et al.,2012).We previously mappedﬁve major gene loci involved in the determination of FSI using bulked segregant analysis in a ‘Jonathan’×‘Golden Delicious’mapping population;these were located on LGs11,12,and13of the female parent‘Jonathan’,and on LG10of the male parent‘Golden Delicious’(Sun et al.,2012). However,we did not obtain any QTLs without linkage maps at that time.In this study,to clarify the genetic relationships among fruit weight,length,diameter,and FSI,and we analyzed the inheritance of these external quality traits,and identiﬁed QTLs associated with them.2.Materials and methods2.1.Plant materialsThe apple cultivars‘Jonathan’(J)and‘Golden Delicious’(G),with ‘Jonathan’as the female parent were crossed in spring2002at the Changli Institute of Pomology(Hebei Province,China)to obtain hybrid progeny.Seedlings were planted in2003at a density of one per0.5m×2m plot,resulting in a J×G F1population of1733 seedlings.After planting,the seedlings were subjected to conven-tionalﬁeld management and pest control procedures(Sun et al., 2012).2.2.PhenotypingApples sufﬁcient for phenotyping were harvested in1162 seedlings in2008.Due to alternate bearing,ripening fruit from971 seedlings were collected in2009.A vernier caliper was used for the measurements of fruit diameter(D)and fruit length(L).The phenotypic value used for further analysis was represented by the average values of at leastﬁve apples per seedling each year.FSI was calculated using the formula FSI=L/D.Fruit size was recorded as the average fruit weight,and the phenotypic data of fruit size were the average values ofﬁve apples,which were determined by weighing the fruit on an analytical balance.2.3.Inheritance analysisTo evaluate the quality of phenotypic data to obtain reliable results of QTL identiﬁcation,data of fruit length and fruit diame-ter were subjected to analysis of variance(ANOVA,F-test)using Microsoft Excel2003with30randomly selected seedlings,which bear sufﬁcient amounts of fruit(n=10apples per plant)in both 2008and2009.The correlations of fruit length,diameter,shape, and size were analyzed using data collected from983seedlings in 2008and from789seedlings in2009.Inheritance was analyzed using frequency-distribution analysis,Shapiro–Wilk tests(SPSS v.12.0;SPSS Inc.,Chicago,IL,USA),and chi-square tests(Microsoft Excel2003).This protocol has been previously described by Sun et al.(2012).Phenotypic variance(S)was deﬁned as the sum of genotypic variance(Sg)and environmental variance(Se).Heritabil-ity was calculated as(S−Se)/S×100%,and S was calculated using the variance among the30seedlings.Environmental variance was represented by the average variance among the10apples from each seedling(Sun et al.,2012).2.4.QTL analysisQTL analysis was performed using our previously published genetic linkage maps(Zhang et al.,2012),which consisted of 242individuals and251simple sequence repeat(SSR)markers. Phenotypic data on fruit length,diameter,FSI and size for the map-ping population(n=242seedlings)were collected in2008(n=144 seedlings)and2009(n=140seedlings).MapQTL 6.0(Van Ooijen et al.,2009)was used to analyze QTLs.Interval mapping was performed,and the genome-and chromosome-wide threshold for QTL signiﬁcance of logarithm of odds(LOD)was calculated by performing1000iterations using the MapQTL Permutation Test.The genome-wide threshold was LOD=2.80at the95%conﬁdence interval.3.Results3.1.Phenotype evaluationThere was signiﬁcant variation in fruit diameter,length,and FSI among the seedlings and between the sampling years,but there were no signiﬁcant differences among apples from individ-ual seedlings(Table1).Unfortunately,ANOVA could not be used for fruit size because phenotypic data were obtained by averaging the weight of10apples from each seedling.Fruit length and diameter were signiﬁcantly correlated(r>0.70) in both2008and2009.FSI was positively correlated with fruit length,and negatively correlated with fruit diameter.The abso-lute values of correlation coefﬁcients between FSI and fruit length were larger than those between FSI and fruit diameter,suggesting that length was a more pronounced trait than diameter.Although both length and diameter were positively correlated with fruit size, the correlation was stronger for diameter,indicating that fruit size128Y.Chang et al./Scientia Horticulturae 174(2014)126–132Fig.1.Frequency distributions of fruit length,diameter,and size (weight)in progenies from the ‘Jonathan’×‘Golden Delicious’hybrid cross.Phenotypic data were collected in 2008and 2009.The parental values are indicated on the ﬁgures with vertical dash lines.(weight)was more a function of diameter than of length.No signif-icant correlation was detected between FSI and fruit size (Table 2).Fruit size,length,and diameter followed normal distribution patterns in both sampling years,and they showed features typical of quantitative traits controlled by polygenes without major gene segregation (Fig.1).The broad-sense heritability of fruit length and diameter were estimated as 91%and 93%,respectively in 2008;and as 82%and 85%in 2009.These values indicated that environmental effects had a greater effect on fruit quality in 2009(Table 3).3.2.QTL analysisNineteen QTLs for fruit size,shape,length,and diameter were identiﬁed at the whole-genome level based on a LOD thresh-old ≥2.80in both sampling years (Table 4).Twenty-six additionalTable 1F -tests of phenotypic traits in apple fruit.VariationTraitYearFF 0.01Seedlings Length 2008106.62* 1.78200947.57* 1.78Diameter2008142.01* 1.78200960.69*1.78ReplicatesLength20080.22 2.4720090.70 2.47Diameter20080.147 2.4720090.542.47YearsLength 56.53* 6.68Diameter23.05*6.68*Signiﬁcant difference at P ≤0.01as determined using Duncan’s test.QTLs were identiﬁed,based on a permutation test at P =0.05,at the single-chromosome-based LOD threshold (Table 4,Fig.2).Of these,eight QTLs related to fruit length were detected in 2008;no QTLs for fruit length were detected in 2009.Eleven and two QTLs for fruit size were identiﬁed in 2008and 2009,respec-tively.Nine QTLs in 2008and two QTLs in 2009for fruit diameter mapped onto the two parental linkage groups.In addition,we also detected seven and six QTLs associated with FSI in 2008and 2009,respectively.For FSI,one QTL,fsij08.11.2/fsij09.11on LG11of the female parent ‘Jonathan,’and one QTL fsig08.15/fsig09.15.1in the male parent ‘Golden Delicious’were observed in both years (Fig.2).Four QTLs for fruit size,four for diameter,and three for length co-localized and clustered on chromosome 8of ‘Golden Delicious.’The fszg08.11.1QTL for fruit size was tightly linked to ﬂg08.11forTable 2Correlations between apple length,diameter,shape index,and size in a ‘Jonathan’×‘Golden Delicious’hybrid population.Fruit traitFruit lengthFruit diameterFruit shape2008Fruit diameter 0.77*Fruit shape 0.48*−0.19*Fruit size0.76*0.87*−0.0332009Fruit diameter 0.76*Fruit shape 0.42*−0.27*Fruit size0.78*0.89*−0.084983seedlings in 2008and 789in 2009were used to analyze the correlations of fruit length,diameter,shape and size (r 0.05=0.0625and r 0.01=0.082in 2008;r 0.05=0.07and r 0.01=0.09in 2009).*Signiﬁcance at P =0.05.Y.Chang et al./Scientia Horticulturae174(2014)126–132129 Table3Estimated heredity parameters for apple length and diameter in a‘Jonathan’×‘Golden Delicious’hybrid population.Trait Year Average±SD(mm)Population variance(S)Genetic variance(Sg)Environmental variance(Se)Heritability(%) Fruit length200858.66±5.48123.41112.4310.9891.10 200952.88±4.5828.3823.24 5.1481.89Fruit diameter200868.70±5.69156.63145.9610.6793.20 200963.27±5.1540.3334.39 5.9485.30length and to fdg08.11for diameter on LG11of‘Golden Delicious’(Table4,Fig.2).The QTL fszj08.15(fruit size)overlappedﬂj08.15 (fruit length)exactly on chromosome15of‘Jonathan’.The fszg08.3 QTL for fruit-size coincided with fdg08.11.3(fruit diameter)and QTL fszj08.5(fruit size),and partially overlapped fdj08.5(fruit diame-ter)on LG5of‘Jonathan’(Table4,Fig.2).For FSI,fsij08.4partially overlappedﬂj08.4(fruit length)on LG4of Jonathan;fsij09.9was co-localized with fdj09.9on LG9;and fsij08.17was closely linked to ﬂj08.17on LG17of‘Jonathan’(Table4,Fig.2).4.DiscussionFruit size and shape indices were closely associated with length and diameter,whereas the inheritance of fruit size,shape,length, and diameter differed.The normal distribution of phenotypic traits suggests that apple length,diameter,and size are under polygenetic control.However,variation in FSI is associated with segregation in both major genes and polygenes,and the heritability of major genes was found to be as high as75%(Sun et al.,2012).Table4Quantitative trait loci(QTLs)and mapping information for apple size,shape,length,and diameter in segregated progeny of‘Jonathan’×‘Golden Delicious’.Trait Year QTL LG Location Nearest marker LOD Contribution to totalvariance(%)Fruit length2008ﬂj08.15J150.000WBGCAS50 3.5010.10ﬂj08.17J17-20.000NZmsEB137525 2.337.60ﬂj08.4J40.000Hi23g08 2.01 6.20ﬂj08.8J871.700Hi23g12 1.797.30ﬂg08.8.1G869.141H20b03 3.9812.10ﬂg08.8.2G837.552BACSSR46 3.0411.80ﬂg08.8.3G830.644CTG1069672 3.3013.00ﬂg08.11G1116.788CH05c02 2.347.70Fruit diameter2008fdj08.5J591.033NZmsCN898349 2.809.20fdj08.13J1321.582CTG1075622 2.087.20fdg08.2G258.212CH03d10 2.387.30fdg08.3G382.408WBGCAS27 2.258.10fdg08.8.1G868.660Hi20b03 3.1710.1fdg08.8.2G853.250CH05a02 3.0212.5fdg08.8.3G837.552BACSSR46 2.8510.90fdg08.8.4G830.644CTG1069672 3.0311.30fdg08.11G1120.788BACSSR10 2.539.202009fdj09.9J927.391CTG1067792 2.739.10fdg09.4G4 5.000CH01b01b 1.80 6.30Fruit shape index2008fsij08.4J40.000Hi23g08 2.878.40fsij08.17J17-2 5.000CN938125 1.91 6.20fsig08.15G15-1 1.000CH02c09 2.598.00fsij08.11.1J1114.813Hi23d02 4.0012.80fsij08.11.2J117.371CH02d12 3.4210.30fsij08.11.3J11 3.000CH02d08 3.7613.70fsij08.5J57.000CN881672 1.817.702009fsij09.9J924.391CTG1067792 2.659.20fsij09.13J1332.087CTG1075622 2.2110.00fsij09.7J715.069CTG1060504 1.817.20fsig09.15.1G15-1 3.000CH02c09 1.95 6.70fsig09.15.2G15-151.249NZmsEB117266 1.85 5.50fsij09.11J117.371CH02d12 4.0210.20Fruit size2008fszg08.8.1G869.141Hi20b03 4.2912.80fszg08.8.2G851.250CH04g12 3.0712.60fszg08.8.3G837.552BACSSR46 3.1311.50fszg08.8.4G828.644CTG1069672 3.3112.60fszg08.11.1G1124.788BACSSR10 2.979.70fszg08.11.2G119.930CH04a12 2.447.30fszg08.11.3G110.000CH02d08 2.147.10fszj08.5J595.033Hi02a03 2.517.7fszj08.15J150WBGCAS50 2.227.2fszj08.12J1257.944CH03c02 2.019.6fszg08.3G384.408WBGCAS27 1.72 6.42009fszg09.12G1245.311WBGCAS37 2.02 6.7fszg09.14G1494.33NZmsEB146613 1.858.9LG:linkage group;LOD:logarithm of odds.QTLs detected at whole-genome LOD threshold≥2.8are indicated in bold fonts.130Y.Chang et al./Scientia Horticulturae 174(2014)126–132Fig.2.Internal mapping of quantitative trait loci (QTLs)for fruit length,diameter,shape index (FSI),and size using the ‘Jonathan’×‘Golden Delicious’hybrid population.The letters J and G on the top of the linkage maps represent the maternal parent ‘Jonathan’and pollen parent ‘Golden Delicious’,respectively.The number following J and G indicates the number of linkage groups.Homologs between parents on corresponding linkage groups (LGs)are joined to each other with solid black lines.The solid color bars indicate the QTLs identiﬁed on the most likely position of the linkage groups,while the thin lines represent the conﬁdence interval at the 95%level.QTLs for fruit length,diameter,size,and FSI are marked by the black,blue,red,and yellow color bars,respectively.F11-1and F11-2,on LG11of ‘Jonathan’,represent the two major gene loci for FSI detected by Sun et al.(2012).(For interpretation of the references to color in this legend,the reader is referred to the web version of the article.)Our ﬁndings contrasted with previous reports that apple fruit size is a quantitative trait with relatively low heritability (0.33–0.61)(Durel et al.,1997;Oraguzie et al.,2001;Alspach and Oraguzie,2002).The heritability of fruit length and diameter was relatively high (82–93%)during the two years of evaluation.Both FSI and fruit size correlated with fruit length and diameter.QTLs for closely correlated traits should map to the same or simi-lar positions (Paterson et al.,1991;Kenis et al.,2008).Thus,QTLs associated with FSI or fruit size,at least in part,should overlap or be linked to those for fruit length and diameter.Indeed,the three QTLs for fruit size (fszg08.8.1,fszg08.8.3,and fszg08.8.4)completely overlapped QTLs for fruit length (ﬂg08.8.1,ﬂg08.8.3,and ﬂg08.8.4),and those for fruit diameter (fdg08.8.1,fdg08.8.3,and fdg08.8.4).In ‘Telamon’and ‘Braeburn’progeny,QTLs for apple weight,height,and diameter on LG17partially overlapped with QTLs for fruit height and diameter on LG2.Furthermore,year-stable QTLs for fruit weight and diameter overlapped on LG10of the two par-ents (Kenis et al.,2008).Similarly,the QTL for FSI (fsij08.4)precisely overlapped the one for fruit length (ﬂj08.4),whereas fsij09.9and fsij08.17for FSI were closely linked to fdj09.9and ﬂj08.17,respec-tively.These co-localizations conﬁrmed the correlation analysis that indicated that fruit length strongly affects FSI.In our hybrid population,QTLs for fruit length (on LGs 15and 17)and diameter(on LGs 2,5,and 9)were located on the same LGs as QTLs in the ‘Telamon’×‘Braeburn’cross (Kenis et al.,2008).Using two map-ping populations,Devoghalaere et al.(2012)identiﬁed six QTLs for fruit size,on LGs 5,8,11,15,16,and 17;of these,QTLs on LGs 8and 15were conserved across both populations.In hybrid populations derived from European and Chinese pears,QTLs for FSI,weight,and length co-localized on LG8;interestingly,some QTLs clustered on LG7of the female parent (Zhang et al.,2013).However,we did not detect signiﬁcant correlations between FSI and fruit size.Thus,the QTLs for these traits did not map close to each other on the same chromosomes.Rather,QTLs for FSI over-lapped with or were linked to QTLs for fruit length and diameter on chromosomes that were not linked to fruit size,thus demonstrat-ing that FSI and fruit size are controlled by different genes.Such independent genetic control differs fundamentally from other fruit species,such as pear (Zhang et al.,2013).In muskmelon (Cucumis melo L.),the major QTL for fruit shape (fs2.2)is co-localized with a major gene (andromonoecious );this effect is detectable in com-parisons of ovary and fruit length,but not ovary and fruit width (Périn et al.,2002).Another major QTL for fruit shape,fs12.1,co-segregates with another major gene,pentamerous ,and this effect is detectable in comparisons of ovary and fruit width,but not ovary and fruit length (Périn et al.,2002).Y.Chang et al./Scientia Horticulturae174(2014)126–132131We observed a signiﬁcant correlation between fruit length and diameter,and a close relationship between fruit diameter and size. Four QTLs for fruit diameter,compared with only one QTL for fruit length,co-segregated with or closely linked to QTLs for fruit size. Four QTLs also contributed simultaneously to fruit size,length, and diameter.Instability of QTLs between different years of detec-tion has been reported for many species(Liebhard et al.,2003; Zhang et al.,2013).However,only two QTLs,fsij08.11.2/fsij09.11 and fsig08.15/fsig09.15.1,were stable across the two-year study.The variation in fruit length and diameter between the sampling years indicates that environmental effects or genotype–environment interactions affect the robustness of QTLs between years.Kenis et al.(2008)also observed that QTLs for fruit weight,diameter,and height differed among years.QTL-mapping software provides a powerful tool for detecting major genes for qualitative and quantitative traits(Jones et al., 1997).Our previous study used the same data sets to identifyﬁve major gene loci involved in apple FSI(Sun et al.,2012).Of theseﬁve loci,F11-1(Fig.2),ﬂanked by CH02d08and CH04a12,mapped to the same region as the year-stable QTL fsij08.11.2/fsij09.11at7.371 cM on chromosome11of the female parent‘Jonathan’,closest to CH02d12.The major gene locus F13was located in the same region as the QTL fsij09.13(Sun et al.,2012).In the apple genome,more than10genes related to fruit growth and development,including genes involved in cell division and auxin signaling,are scattered in the region of CH02d12,at7.371 cM on LG11.An auxin response factor gene,ARF106,which modu-lates cell division and expansion,is co-localized with a stable QTL for fruit weight in duplicated regions on LGs8and15of the apple genome(Devoghalaere et al.,2012).In conclusion,45QTLs for apple fruit size,shape,length,and diameter were identiﬁed from a‘Jonathan’×’Golden Delicious’population.Of the19QTLs for fruit length and diameter,12over-lapped with or tightly linked to QTLs for fruit size,and another three co-localized with QTLs for fruit shape.None of the QTLs for fruit size mapped to the same region as QTLs for fruit shape,indicating that fruit size and shape are under independent genetic control.AcknowledgmentsThis work was supported by the Hi-Tech Research and Devel-opment(863)Program of China(2011AA001204);National Special Funds for Scientiﬁc Research on Public Causes(Agriculture)Project 200903044;Modern Agricultural Industry Technology System (Apple)(CARS-28);and Key Laboratory of Biology and Genetic Improvement of Horticultural Crops(Nutrition and Physiology), Ministry of Agriculture,P.R.China.Appendix A.Supplementary dataSupplementary data associated with this article can be found,in the online version,at /10.1016/j.scienta. 2014.05.019.ReferencesAlspach,P.A.,Oraguzie,N.C.,2002.Estimation of genetic parameters of apple(Malus domestica)fruit quality from open-pollinated families.New Zeal.J.Crop Hortic.Sci.30,219–228.Baldi,P.,Patocchi,A.,Zini,E.,Toller,C.,Velasco,R.,Komjanc,M.,2004.Cloning and linkage mapping of resistance gene homologues in apple.Theor.Appl.Genet.109,231–239.Brewer,M.T.,Lang,L.,Fujimura,K.,Dujmovic,N.,Gray,S.,Van der Knaap,E.,2006.Development of a controlled vocabulary and software application to analyze fruit shape variation in tomato and other plant species.Plant Physiol.141,15–25. 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Dash,M.,Malladi,A.,2012.The AINTEGUMENTA genes,MdANT1and MdANT2,are associated with the regulation of cell production during fruit growth in apple (Malus×domestica Borkh.).BMC Plant Biol.12,98.Denne,M.P.,1963.Fruit development and some tree factors affecting it.New Zeal.J.Bot.1,265–294.Devoghalaere,F.,Doucen,T.,Guitton,B.,Keeling,J.,Payne,W.,Ling,T.J.,Ross,J.J., Hallett,I.C.,Gunaseelan,K.,Dayatilake,G.A.,Diak,R.,Breen,K.C.,Tustin,D.S., Costes,E.,Chagne,D.,Schaffer,R.J.,David,K.M.,2012.A genomics approach to understanding the role of auxin in apple(Malus×domestica)fruit size control.BMC Plant Biol.12,7.Durel,C.E.,Laurens,F.,Fouillet,A.,Lespinasse,Y.,1998.Utilization of pedigree infor-mation to estimate genetic parameters from large unbalanced data sets in apple.Theor.Appl.Genet.96,1077–1085.Eccher,T.,Boffelli,G.,1981.Effect of dose and time of application of GA on russeting fruit set and shape of Golden Delicious apple.Sci.Hortic.14, 307–314.Fernández-Fernández,F.,Evans,K.M.,Clarke,J.B.,Govan,C.L.,James,C.M.,Mariˇc,S., Tobutt,K.R.,2008.Development of an STS map of an interspeciﬁc progeny of Malus.Tree Genet.Genomics4,469–479.Gao,Z.S.,Van de Weg,W.E.,Schaart,J.G.,Van der Meer,I.M.,Kodde,L.,Laimer,M., Breiteneder,H.,Hoffmann-Sommergruber,K.,Gilissen,L.J.W.J.,2005.Linkage map positions and allelic diversity of two Mal d3(non-speciﬁc lipid transfer protein)genes in the cultivated apple(Malus domestica).Theor.Appl.Genet.110,479–491.Garcia-Martinez,J.L.,Sponsel,V.M.,Gaskin,P.,1987.Gibberellins in developing fruits of Pisum sativum cv.Alaska:studies on their role in pod growth and seed devel-opment.Planta170,130–137.Greene,D.W.,1986.Effect of paclobutrazol and analogs on growth yield,fruit quality, and storage of‘Delicious’apples.J.Am.Soc.Hortic.Sci.111,328–332. Gonzalo,M.J.,Brewer,M.T.,Anderson,C.,Sullivan,D.,Gray,S.,Van der Knaap,E.,2009.Tomato fruit shape analysis using morphometric and morphologyattributes implemented in tomato analyzer software program.J.Am.Soc.Hortic.Sci.134,77–87.Hemmat,M.,Weeden,N.F.,Manganaris,A.G.,Lawson,D.M.,1994.Molecular marker linkage map for apple.J.Hered.85,4–11.Jones,N.,Ougham,H.,Thomas,H.,1997.Markers and mapping:we are all geneticists now.New Phytol.137,165–177.Kenis,K.,Keulemans,J.,Davey,M.W.,2008.Identiﬁcation and stability of QTLs for fruit quality traits in apple.Tree Genet.Genomes4,647–661.Lerceteau-Köhler,E.,Moing,A.,Guérin,G.,Renaud,C.,Petit,A.,Rothan,C.,Denoyes,B.,2012.Genetic dissection of fruit quality traits in the octoploid cultivatedstrawberry highlights the role of homoeo-QTL in their control.Theor.Appl.Genet.124,1059–1077.Liebhard,R.,Gianfranceschi,L.,Koller,B.,Ryder,C.D.,Tarchini,R.,Van De Weg,E., Gessler,C.,2002.Development and characterisation of140new microsatellites in apple(Malus×domestica Borkh.).Mol.Breeding10,217–241.Liebhard,R.,Kellerhals,M.,Pfammatter,W.,Jertmini,M.,Gessler,C.,2003.Mapping quantitative physiological traits in apple(Malus×domestica Borkh.).Plant Mol.Biol.52,511–526.Maliepaard,C.,Alston,F.H.,Van Arkel,G.,Brown,L.M.,Chevreau,E.,Dunemann,F., Evans,K.M.,Gardiner,S.,Guilford,P.,Van Heusden,A.W.,Janse,J.,Laurens,F., Lynn,J.R.,Manganaris,A.G.,Den Nijs,A.P.M.,Periam,N.,Rikkerink,E.,Roche, P.,Ryder,C.,Sansavini,S.,Schmidt,H.,Tartarini,S.,Verhaegh,J.J.,Vrielink-van Ginkel,M.,King,G.J.,1998.Aligning male and female linkage maps of apple (Malus pumila Mill.)using multi-allelic markers.Theor.Appl.Genet.97,60–73. Meneses,C.,Orellana,A.,ing genomics to improve fruit quality.Biol.Res.46,347–352.Oraguzie,N.C.,Hofstee,M.E.,Brewer,L.R.,Howard,C.,2001.Estimation of genetic parameters in a recurrent selection program in apple.Euphytica118,29–37. Paterson,A.H.,Damon,S.,Hewitt,J.D.,Zamir,D.,Rabinowitch,H.D.,Lincoln,S.E., Lander,E.S.,Tanksley,S.D.,1991.Mendelian factors underlying quantitative traits in tomato:comparison across species,generations,and environments.Genetics127,181–197.Périn,C.,Hagen,L.S.,Giovinazzo,N.,Besombes,D.,Dogimont,C.,Pitrat,M.,2002.Genetic control of fruit shape acts prior to anthesis in melon(Cucumis melo L.).Mol.Genet.Genomics266,933–941.Sadrnia,H.,Rajabipour,A.,Jafary,A.,Javadi,A.,Mostoﬁ,A.,2007.Classiﬁcation and analysis of fruit shapes in long type watermelon using image processing.Int.J.Agric.Biol.9,68–70.Shaw,J.K.,1914.A study in variation in apples.Mass.Agric.Exp.Stn.Bull.149,21–36. Silfverberg-Dilworth,E.,Matasci,C.L.,Van de Weg,W.E.,Van Kaauwen,M.P.W., Walser,M.,Kodde,L.P.,Soglio,V.,Gianfranceschi,L.,Durel, C.E.,Costa, F., Yamamoto,T.,Koller,B.,Gessler,C.,Patocchi,A.,2006.Microsatellite markers spanning the apple(Malus×domestica Borkh.)genome.Tree Genet.Genomes2, 202–224.。

苹果PGIP基因的克隆及其在大肠杆菌中的表达

苹果PGIP基因的克隆及其在大肠杆菌中的表达熊帅;张军科;谌悦【摘要】[目的]从富士苹果幼果果实中克隆多聚半乳糖醛酸酶抑制蛋白(PGIP)基因,并进行原核表达,为进一步研究PGIP的生物学功能奠定基础.[方法]根据Genbank 中已经发表的苹果PGIP基因序列设计引物,采用RT-PCR从苹果果实中扩增PGIP 基因cDNA,回收目的基因片段并连接到pMD18-T载体,鉴定后进行测序.然后将PGIP全长cDNA和去信号肽的cDNA导入到pET-32a(+)表达载体中,分别获得融合表达质粒pET-PGIP和pET-PGIP-X,将其分别转化大肠杆菌BL21感受态细胞,用不同终浓度IPTG进行诱导表达,收集表达产物并进行SDS-PAGE电泳.对pET-PGIP-X基因工程菌表达产物的可溶性进行检测.[结果]苹果PGIP cDNA序列长为1 091 bp,编码区为993 bp,可编码330个氨基酸残基,将其命名为MdPGIP;重组表达质粒pET-PGIP和pET-PGIP.X在宿主大肠杆菌中分别表达出分子质量约49.6和46.1 ku的融合蛋白,pET-PGIP-X表达产物以包涵体的形式存在.[结论]成功克隆了苹果PGIP基因,并在大肠杆菌中获得高效表达.【期刊名称】《西北农林科技大学学报（自然科学版）》【年(卷),期】2010(038)002【总页数】7页(P123-128,134)【关键词】苹果;多聚半乳糖醛酸酶抑制蛋白;基因克隆;内源多聚半乳糖醛酸酶【作者】熊帅;张军科;谌悦【作者单位】西北农林科技大学,园艺学院,农业部西北园艺植物种质资源利用重点开放实验室,陕西,杨凌,712100;西北农林科技大学,园艺学院,农业部西北园艺植物种质资源利用重点开放实验室,陕西,杨凌,712100;西北农林科技大学,园艺学院,农业部西北园艺植物种质资源利用重点开放实验室,陕西,杨凌,712100【正文语种】中文【中图分类】S661.1植物细胞壁可以有效地阻碍植物病原真菌的侵染，但植物病原真菌可分泌一系列的酶来降解植物细胞壁。

苹果“大群体”杂交后代果实性状评价方法的优化

摘要摘要苹果（Malus domestica Borkh.）新品种选育的方法与途径较多，其中杂交育种是最为有效的方法。

目前，在育种研究中“少组合、大群体”的育种策略被广泛应用，苹果育种群体普遍较大。

相较于小群体而言，大群体育种的工作量增加数倍，其中复选阶段果实性状的评价工作难度最大。

为提高育种研究的效率，本研究选用‘富士’×‘嘎拉’、‘富士’×‘金冠’、‘富士’×‘秦冠’和‘富士’×‘粉红女士’4个杂交组合的部分杂交后代群体为试材，于2018年和2019年的8月~11月，采用感官评价和仪器测定相结合的方式，对复选过程中的果实品质性状进行分级鉴评。

按三级选择淘汰方法确定评价指标，分批淘汰杂种后代个体，并统计淘汰比率。

旨在简化果实性状评价指标，优化果实性状评价方法，为简化苹果“大群体”杂交育种程序、提高选择效率提供依据。

取得的主要研究结果如下：1.对4个杂交组合杂种F1代的果实外观性状和内在品质性状进行感官评价和测定分析。

结果表明，果形、颜色、果锈、果肉质地、果肉粗细、汁液多少等感官指标在4个组合的杂交后代中分离广泛，单果重、可溶性固形物、果肉硬度等理化指标，在4个组合的杂交后代中均呈现两端少、中间多的分布状态，属于表型连续变异的数量性状，但颜色不符合数量性状遗传特点。

在连续两年的调查期间，各性状的遗传变异稳定。

2.果实性状评价方法的优化。

对评价指标进行整理分析，并建立了果实性状三级评价体系：一级评价以果锈、异味作为评价指标，主要淘汰果实有明显缺陷的植株；二级评价以光洁度、着色程度、质地、风味作为评价指标，香气为辅助选择指标，淘汰果实主要品质性状表现较差的植株；三级评价以感官综合评价为主，同时结合果肉硬度和可溶性固形物两个理化指标，对淘汰后选留的优系进行同熟期对比优选。

3.将杂交后代的复选淘汰率阈值定为95%。

利用2018年、2019年的杂交后代果实性状调查数据，对评价指标的合理性和分级评价方法的可行性进行验证。

苹果多酚氧化酶特性及无硫护色脱水研究

334刘树兴，王乐*（陕西科技大学生命与工程学院，陕西西安710021）摘要：脱水苹果片在加工过程中极易发生褐变。

在对苹果多酚氧化酶特性进行分析的基础上，通过设计正交实验得到最佳无硫护色剂组合，并初步确定真空干燥的条件。

结果表明，苹果多酚氧化酶的最适pH 为6.0，最适温度为36ħ，100ħ热烫60s 可使其完全失活。

苹果片的最佳无硫护色剂组合为0.6%柠檬酸、0.06%L－半胱氨酸、1.5%氯化钠，在真空度为0.09MPa ，温度为50ħ的条件下，可得到色泽较好的苹果片。

关键词：脱水苹果片，多酚氧化酶，无硫护色，真空干燥Characteristics of polyphenol oxidase in apple andnon －sulfur prevention of discoloration in dried apple slice processingLIU Shu －xing ，WANG Le *（College of Life Science ＆Engineering ，Shaanxi University of Science ＆Technology ，Xi ’an 710021，China ）Abstract ：Browning occured very easily during the procession of dried apple slice .In this study ，the characteristics of polyphenol oxidase in apple were investigated .By an orthogonal experiment of L 9（34），a kind of mixed soaking solution which was safe and effective for color protection was obtained .The results showed that polyphenol oxidase in apple was actively at pH 6.0and 36ħ，and the activity was almost lost in condition of 100ħ，60s .After the apple were soaked in the mixed solution which consisted of 0.6%citric acid ，0.06%L －cys and 1.5%NaCl ，and dried at 50ħ，0.09MPa ，the color of the dried apple slice was good .Key words ：dried apple slice ；polyphenol oxidase ；non －sulfites color protection ；vacuum drying 中图分类号：TS255.1文献标识码：B文章编号：1002－0306（2011）03－0334－03收稿日期：2010－03－19*通讯联系人作者简介：刘树兴（1962－），男，教授，硕士，研究方向：食品加工与食品添加剂研制。

21世纪是生命科学的世纪20世纪后叶分子生物学的突破性...

第一章绪论一简答题1. 21世纪是生命科学的世纪。

20世纪后叶分子生物学的突破性成就，使生命科学在自然科学中的位置起了革命性的变化。

试阐述分子生物学研究领域的三大基本原则，三大支撑学科和研究的三大主要领域？答案：（1）研究领域的三大基本原则：构成生物大分子的单体是相同的；生物遗传信息表达的中心法则相同；生物大分子单体的排列（核苷酸，氨基酸）导致了生物的特异性。

（2）三大支撑学科：细胞学，遗传学和生物化学。

（3）研究的三大主要领域：主要研究生物大分子结构与功能的相互关系，其中包括DNA和蛋白质之间的相互作用；激素和受体之间的相互作用；酶和底物之间的相互作用。

2. 分子生物学的概念是什么？答案：有人把它定义得很广：从分子的形式来研究生物现象的学科。

但是这个定义使分子生物学难以和生物化学区分开来。

另一个定义要严格一些，因此更加有用：从分子水平来研究基因结构和功能。

从分子角度来解释基因的结构和活性是本书的主要内容。

3 二十一世纪生物学的新热点及领域是什么？答案：结构生物学是当前分子生物学中的一个重要前沿学科，它是在分子层次上从结构角度特别是从三维结构的角度来研究和阐明当前生物学中各个前沿领域的重要学科问题，是一个包括生物学、物理学、化学和计算数学等多学科交叉的，以结构（特别是三维结构）测定为手段，以结构与功能关系研究为内容，以阐明生物学功能机制为目的的前沿学科。

这门学科的核心内容是蛋白质及其复合物、组装体和由此形成的细胞各类组分的三维结构、运动和相互作用，以及它们与正常生物学功能和异常病理现象的关系。

分子发育生物学也是当前分子生物学中的一个重要前沿学科。

人类基因组计划，被称为“21世纪生命科学的敲门砖”。

“人类基因组计划”以及“后基因组计划”的全面展开将进入从分子水平阐明生命活动本质的辉煌时代。

目前正迅速发展的生物信息学，被称为“21世纪生命科学迅速发展的推动力”。

尤应指出，建立在生物信息基础上的生物工程制药产业，在21世纪将逐步成为最为重要的新兴产业；从单基因病和多基因病研究现状可以看出，这两种疾病的诊断和治疗在21世纪将取得不同程度的重大进展；遗传信息的进化将成为分子生物学的中心内容”的观点认为，随着人类基因组和许多模式生物基因组序列的测定，通过比较研究，人类将在基因组上读到生物进化的历史，使人类对生物进化的认识从表面深入到本质；研究发育生物学的时机已经成熟。

Henkel Ireland Limited 99C 362 5C 产品说明书

Safety Data Sheet according to (EC) No 1907/2006Page 1 of 8sds no. : 221815V004.0 99C 362 5CRevision: 14.05.2012printing date: 23.07.2012SECTION 1: Identification of the substance/mixture and of the company/undertaking1.1. Product identifier99C 362 5C1.2. Relevant identified uses of the substance or mixture and uses advised againstIntended use:Solder Wire1.3. Details of the supplier of the safety data sheetHenkel Ireland LimitedProduct Safety & Regulatory AffairsTallaght Business Park, WhitestownDublin 24IrelandPhone: +353 (14046444)Fax-no.: +353 (14519926)*****************************.com1.4. Emergency telephone number24 Hours Emergency Tel: +44 (0)1442 278497SECTION 2: Hazards identification2.1. Classification of the substance or mixtureClassification (DPD):SensitizingR43 May cause sensitisation by skin contact.2.2. Label elementsLabel elements (DPD):Xi - IrritantRisk phrases:R43 May cause sensitisation by skin contact.Safety phrases:S24 Avoid contact with skin.S37 Wear suitable gloves.S23 Do not breathe fumes.Contains:Rosin2.3. Other hazardsFlux fumes emitted during reflow will irritate the nose and throat and may cause an asthmatic type reaction.Regulations forbid the use of lead solder in any private or public drinking water supply system.SECTION 3: Composition/information on ingredients Declaration of the ingredients according to CLP (EC) No 1272/2008:Hazardous componentsCAS-No.EC NumberREACH-Reg No.content ClassificationTin 7440-31-5231-141-801-2119486474-28>= 90- < 100%Copper Metal7440-50-8231-159-6 >= 0,1- < 1%Rosin 8050-09-7 232-475-7 >= 1- < 5% Skinsensitizer1H317For full text of the H - statements and other abbreviations see section 16 "Other information". Substances without classification may have community workplace exposure limits available. Declaration of ingredients according to DPD (EC) No 1999/45:Hazardous componentsCAS-No.EC NumberREACH-Reg No.content ClassificationTin 7440-31-5231-141-801-2119486474-28>= 90 - < 100 %Copper Metal7440-50-8231-159-6 >= 0,1 - < 1 %Rosin 8050-09-7 232-475-7 >= 1 - < 5 %R43For full text of the R-Phrases indicated by codes see section 16 'Other Information'. Substances without classification may have community workplace exposure limits available.SECTION 4: First aid measures4.1. Description of first aid measuresInhalation:Move to fresh air. If symptoms persist, seek medical advice.Skin contact:Rinse with running water and soap.Obtain medical attention if irritation persists.Eye contact:Flush eyes with plenty of water for at least 5 minutes. If irritation persists seek medical attention.Ingestion:Do not induce vomiting.Seek medical advice.4.2. Most important symptoms and effects, both acute and delayedFlux fumes may irritate the nose, throat and lungs and may after prolonged/repeated exposure give an allergic reaction (asthma).SKIN: Rash, Urticaria.4.3. Indication of any immediate medical attention and special treatment neededSee section: Description of first aid measuresSECTION 5: Firefighting measures5.1. Extinguishing mediaSuitable extinguishing media:Carbon dioxide, foam, powderFine water sprayExtinguishing media which must not be used for safety reasons:Do not use water on fires where molten metal is present.5.2. Special hazards arising from the substance or mixtureHigh temperatures may produce heavy metal dust, fumes or vapours.The flux medium will give rise to irritating fumes.5.3. Advice for firefightersWear self-contained breathing apparatus.SECTION 6: Accidental release measures6.1. Personal precautions, protective equipment and emergency proceduresWear protective equipment.6.2. Environmental precautionsDo not empty into drains / surface water / ground water.6.3. Methods and material for containment and cleaning upScrape up spilled material and place in a closed container for disposal.6.4. Reference to other sectionsSee advice in chapter 8SECTION 7: Handling and storage7.1. Precautions for safe handlingExtraction is necessary to remove fumes evolved during reflow.When using do not eat, drink or smoke.Wash hands before breaks and immediately after handling the product.Avoid breathing fumes given out during soldering.See advice in chapter 8Hygiene measures:Good industrial hygiene practices should be observed.Do not eat, drink or smoke while working.After handling solder wash hands with soap and water before eating, drinking or smoking.7.2. Conditions for safe storage, including any incompatibilitiesEnsure good ventilation/extraction.Store in a cool, dry place.7.3. Specific end use(s)Solder WireSECTION 8: Exposure controls/personal protection8.1. Control parametersValid forGreat BritainIngredient ppm mg/m3Type Category RemarksTIN (INORGANIC COMPOUNDS AS SN) 7440-31-5 2 Time Weighted Average(TWA):Indicative ECTLVROSIN-BASED SOLDER FLUX FUME 8050-09-7 0,05 Time Weighted Average(TWA):EH40 WELROSIN-BASED SOLDER FLUX FUME 8050-09-7 0,15 Short Term ExposureLimit (STEL):EH40 WELColophony (Rosin) and derivatives: Rosin-based flux fume as total resin acids.8.2. Exposure controls:Engineering controls:Extraction is necessary to remove fumes evolved during reflow.Where reasonably practicable this should be achieved by the use of local exhaust ventilation and good general extraction.Ensure good ventilation/extraction.Respiratory protection:Ensure adequate ventilation.An approved mask or respirator fitted with an organic vapour cartridge should be worn if the product is used in a poorly ventilated areaIn case of aerosol formation, we recommend wearing of appropriate respiratory protection equipment with ABEK P2 filter.This recommendation should be matched to local conditions.Hand protection:Please note that in practice the working life of chemical resistant gloves may be considerably reduced as a result of many influencing factors (e.g. temperature). Suitable risk assessment should be carried out by the end user. If signs of wear and tear are noticed then the gloves should be replaced.The use of chemical resistant gloves such as Nitrile are recommended.Eye protection:Safety glasses with sideshields or chemical safety goggles should be worn if there is a risk of splashing.Skin protection:Wear suitable protective clothing.SECTION 9: Physical and chemical properties9.1. Information on basic physical and chemical propertiesAppearance solidgreyOdor NoneapplicablepH notInitial boiling point No data available / Not applicableFlash point NoneDecomposition temperature No data available / Not applicableVapour pressure not applicable7,3 g/cm3Density(25 °C (77 °F))Bulk density No data available / Not applicableViscosity No data available / Not applicableViscosity (kinematic) No data available / Not applicableExplosive properties No data available / Not applicableInsolubleSolubility (qualitative)(Solvent: Water)Solidification temperature No data available / Not applicableMelting point227 °C (440.6 °F)Flammability No data available / Not applicableAuto-ignition temperature No data available / Not applicableExplosive limits No data available / Not applicablePartition coefficient: n-octanol/water Not applicableEvaporation rate No data available / Not applicableVapor density No data available / Not applicableOxidising properties No data available / Not applicable9.2. Other informationNo data available / Not applicableSECTION 10: Stability and reactivity10.1. ReactivitySolder alloy will react with concentrated nitric acid to produce toxic fumes of nitrogen oxides.10.2. Chemical stabilityStable under recommended storage conditions.10.3. Possibility of hazardous reactionsSee section reactivity10.4. Conditions to avoidNo decomposition if stored and applied as directed.10.5. Incompatible materialsNone if used properly.10.6. Hazardous decomposition productsThermal decomposition can lead to release of irritating gases and vapors.SECTION 11: Toxicological information11.1. Information on toxicological effectsGeneral toxicological information:The preparation is classified based on the conventional method outlined in Article 6(1)(a) of Directive 1999/45/EC. Relevant available health/ecological information for the substances listed under Section 3 is provided in the following.Oral toxicity:This material is considered to have low toxicity if swallowed.Inhalative toxicity:Fumes evolved at soldering temperatures will irritate the nose, throat and lungs. Prolonged or repeated exposure to flux fumes may result in sensitisation in sensitive workers.Dermal toxicity:This product is considered to have low dermal toxicity.Skin irritation:Fumes emitted during soldering may irritate the skin.Eye irritation:Fumes emitted during soldering may irritate the eyes.Sensitizing:May cause sensitization by skin contact.Skin corrosion/irritation:Hazardous components CAS-No. Result ExposuretimeSpecies MethodCopper Metal7440-50-8irritating Serious eye damage/irritation:Hazardous components CAS-No. Result ExposuretimeSpecies MethodCopper Metal7440-50-8irritatingSECTION 12: Ecological informationGeneral ecological information:Do not empty into drains / surface water / ground water.The preparation is classified based on the conventional method outlined in Article 6(1)(a) of Directive 1999/45/EC. Relevant available health/ecological information for the substances listed under Section 3 is provided in the following.Ecotoxicity:May cause long-term adverse effects in the aquatic environment.Mobility:The product is insoluble and sinks in water.Persistence and Biodegradability:The product is not biodegradable.Bioaccumulative potential:Octanol/Water distribution coefficient: Not applicable12.1. ToxicityHazardous componentsCAS-No. ValuetypeValue AcuteToxicityStudyExposuretimeSpecies MethodCopper Metal 7440-50-8 LC50> 10 mg/l Fish96 h Lepomis macrochirus OECD Guideline203 (Fish, AcuteToxicity Test)Rosin 8050-09-7 LC50> 1.000 mg/l Fish96 h Pimephales promelas OECD Guideline203 (Fish, AcuteToxicity Test)Rosin 8050-09-7 EC50911 mg/l Daphnia48 h Daphnia magna OECD Guideline202 (Daphnia sp.AcuteImmobilisationTest)Rosin 8050-09-7 EC50> 100 mg/l Algae72 h Scenedesmus subspicatus (newname: Desmodesmussubspicatus)12.2. Persistence and degradabilityHazardous componentsCAS-No. Result RouteofapplicationDegradability MethodRosin 8050-09-7aerobic 36 - 46 % OECD Guideline 301 F (ReadyBiodegradability: ManometricRespirometry Test) SECTION 13: Disposal considerations13.1. Waste treatment methodsProduct disposal:Wherever possible unwanted solder alloy should be recycled for recovery of metal.Otherwise dispose of in accordance with local and national regulations.Disposal of uncleaned packages:Dispose of as unused product.Waste code06 04 05 - wastes containing other heavy metalsSECTION 14: Transport informationGeneral information:Not hazardous according to RID, ADR, ADNR, IMDG, IATA-DGR.SECTION 15: Regulatory information15.1. Safety, health and environmental regulations/legislation specific for the substance or mixtureVOC content< 5,0 %National regulations/information (Great Britain):Remarks The Health & Safety at Work Act 1974.The Control of Substances Hazardous to Health Regulations. L5:GeneralApproved Code of Practice to the COSHH Regulations. HS(G)97:A Step by StepGuide to the COSHH Regulations. HS(G)193:COSHH essentials: Easy steps tocontrol chemicals.IND (G)248L:Solder fume and you. IND(G)249L:Controlling health risks fromrosin (colophony) based solder fluxes.SECTION 16: Other informationThe labelling of the product is indicated in Section 2. The full textof all abbreviations indicated by codes in this safety data sheet are as follows:R43 May cause sensitisation by skin contact.H317 May cause an allergic skin reaction.Further information:This information is based on our current level of knowledge and relates to the product in the state in which it is delivered. It is intended to describe our products from the point of view of safety requirements and is not intended to guarantee anyparticular properties.This safety data sheet was prepared in accordance with Council Directive 67/548/EEC and it's subsequent amendments, and Commission Directive 1999/45/EC.。

应用DPPH·法测定苹果提取物的抗氧化能力

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&1! %% # 析 # 国外已有很多的学者用此法研究了物质清除自由基的性质 $ ! !% 苹果果皮中的天然成分及抗氧化能力研究是近年来国外研究的热点问题 $ #具有较强抗氧化能力 % ! " 的果实不会发生冷藏苹果的生理病害 $ " 且苹果具有较高的营养价值" 但采用 c \ \ O! 分析评价苹果水

应用PCR技术快速检测腐烂苹果中扩展青霉菌

应用PCR 技术快速检测腐烂苹果中扩展青霉菌何鸿举1，焦凌霞2，樊明涛1,*，张建兵3，吕丽娟1，刘晓娇1，李亚菲1(1.西北农林科技大学食品科学与工程学院，陕西杨凌 712100；2.河南科技学院食品学院，河南新乡 453003；3.陕西出入境检验检疫局国际旅行卫生保健中心，陕西西安 710068)摘要：利用扩展青霉的多聚半乳糖醛酸酶基因序列，设计一对特异性引物，通过聚合酶链式反应(polymerase chain reaction ，PCR)反应快速检测腐烂苹果中的扩展青霉。

反应的特异性和敏感性以及反应的最适条件等试验的结果表明：该引物具有很高的特异性和灵敏性，只扩增扩展青霉DNA ，而不扩增其他真菌DNA ；菌体的检测限为1.34×103个孢子/mL ，DNA 的检测限为2.40×10-2μg/mL ；反应的最适退火温度范围为54～59℃，最适模板浓度范围为2.40～5.28μmol/L 。

该方法具有简单、快速、特异性好和灵敏性高等特点，可为快速检测腐烂苹果中扩展青霉的实际应用提供借鉴。

关键词：聚合酶链式反应(PCR)检测；扩展青霉；DNA 模板；引物特异性Rapid Detection of Penicillium expansum in Rotten Apples by Polymerase Chain ReactionHE Hong-ju 1，JIAO Ling-xia 2，FAN Ming-tao 1,*，ZHANG Jian-bing 3，LU Li-juan 1，LIU Xiao-jiao 1，LI Ya-fei 1(1. College of Food Science and Engineering, Northwest A & F University, Yangling 712100, China ；2. College of Food Science, Henan Institute of Science and Technology, Xinxiang 453003, China ；3. Shaanxi International Travel Healthcare Center, Shaanxi Entry-Exit Inspection and Quarantine Bureau, Xi ’an 710068, China)Abstract ：In this study, a pair of specific primers was designed according to the polygalacturonase gene of Penicillium expansum to detect Penicillium expansum in rotten apples by polymerase chain reaction (PCR). The results showed that the specificity and sensitivity of the design primer pair were high and only Penicillium expansum DNAs were amplified.The detection limits of Penicillium expansum and DNA were 1.34 × 103 spores/mL and 2.40 × 10-2μg/mL, respectively.The optimal reaction condition was obtained as follows: anneal temperature of 54－59 ℃ and template concentration of 2.40－5.28μg/mL. Owing to its simplicity, rapidity and high specificity and sensitivity, this assay can be used for practical applications.Key words ：PCR(polymerase chain reaction)detection ；Penicillium expansum ；template ；primers ；specificity 中图分类号：Q939.92 文献标识码：A 文章编号：1002-6630(2011)12-0183-05收稿日期：2010-07-11基金项目：教育部博士点基金项目(200807120017)；陕西省科技攻关项目(2007K01-12)作者简介：何鸿举(1983—)，男，硕士研究生，主要从事食品生物技术研究。

苹果多酚对四氯化碳致小鼠急性肝损伤的保护作用

苹果多酚对四氯化碳致小鼠急性肝损伤的保护作用史珅2张泽生1*张民1，2张颖1，2侯冬梅1，2唐小红1，2（1食品营养与安全省部共建教育部重点实验室天津科技大学天津3004572天津科技大学食品工程与生物技术学院天津300457）摘要目的：研究苹果多酚对四氯化碳造成的肝损伤的预防和保护作用。

方法：采用四氯化碳制作ICR 小鼠肝损伤模型，灌胃苹果多酚进行预防，检测血清肝功能主要指标白蛋白、胆碱酯酶、碱性磷酸酶、谷草转氨酶、谷丙转氨酶以及丙二醛和总抗氧化能力，探讨苹果多酚对小鼠急性肝损伤的保护作用。

结果：灌胃苹果多酚，尤其是200mg/kg bw/d 剂量水平，可以抑制或减缓肝损伤，保护肝脏功能。

结论：苹果多酚对肝脏及其功能有保护作用。

关键词苹果；多酚；谷丙转氨酶；谷草转氨酶；四氯化碳文章编号1009-7848（2011）03-0009-05苹果多酚是从苹果果实或皮渣中提取的多元酚类物质的总称，易溶于水和乙醇。

粉末状产品于室温下可保存1年，其性质及生理功能不变[1]。

苹果多酚主要组成包括黄酮醇类、羟基肉桂酸类、儿茶素类及其聚合物、二氢查耳酮类等。

研究表明苹果多酚是安全无毒[2]的，具有降血脂、抗动脉粥样硬化[3~6]、抗炎[7~10]、抗氧化[11~12]、抗癌[13~14]和抗菌[15~17]等活性。

目前对肝损伤的防治仍是一个全球性的严峻课题。

各种肝脏疾病发病机制与治疗药物的筛选，在很大程度上依赖于与人类肝脏疾病病理机制相似的实验动物模型的建立与应用。

四氯化碳肝损伤动物模型是用来评价改善肝细胞损伤药物的经典实验性模型，该模型的肝细胞病理学特征与人类病毒性肝炎极为相似，是目前较为推崇的研究保肝药物作用及机制的病理模型。

本文旨在研究苹果多酚对四氯化碳所致肝损伤的预防与保护作用。

采用ICR 小鼠为实验动物，对口服苹果多酚的小鼠灌胃四氯化碳，制作肝损伤模型，通过检测表征机体肝功能的主要指标，探讨苹果多酚对肝细胞损伤的保护作用。

药物筛选器官芯片流程

药物筛选器官芯片流程Drug screening is a crucial step in the drug discovery process. It involves the use of various methods to identify potential drug candidates that could effectively treat a specific disease. One of the most advanced and promising technologies for drug screening is the use of organ-on-a-chip platforms, also known as organ chips. 药物筛选是药物发现过程中的关键步骤。

它涉及使用各种方法来确定潜在的药物候选者，可以有效地治疗特定疾病。

器官芯片平台，也被称为器官芯片，是药物筛选的先进和有前途的技术之一。

Organ chips are microfluidic cell culture devices that simulate the activities, mechanics, and physiological response of entire organs. They are designed to replicate the functions of specific organs, such as the liver, lung, heart, and kidney, in a controlled environment. 器官芯片是微流体细胞培养装置，模拟整个器官的活动、机械和生理反应。

它们旨在在受控环境中复制特定器官的功能，例如肝脏、肺部、心脏和肾脏。

The use of organ chips in drug screening offers several advantages over traditional cell culture and animal models. Firstly, they provide a more accurate representation of human physiology, allowing forbetter predictions of drug responses and toxicities. Additionally, they enable the study of inter-organ interactions and disease mechanisms, which are crucial for understanding the overall effects of a drug on the human body. 器官芯片在药物筛选中的使用相对于传统的细胞培养和动物模型具有几个优点。

pPIC3.5K

pPIC3.5K/pAO815Version D03200225-0156pPIC3.5K/pAO815Pichia vectors for multicopy integration and intracellular expressionCatalog nos. V173-20, V177-20, V180-20tech_service@iiINDIVIDUAL PICHIA EXPRESSION KIT LICENSE AGREEMENTThe Pichia Expression Kit is based on the yeast Pichia pastoris. Pichia pastoris was developed into an expression system by scientists at Salk Institute Biotechnology/Industry Associates (SIBIA) for high-level expression of recombinant proteins. All patents for Pichia pastoris and licenses for its use as an expression system are owned by Research Corporation Technologies, Inc. Tucson, Arizona. Invitrogen has an exclusive license to sell the Pichia Expression Kit to scientists for research purposes only, under the terms described below. Use of Pichia pastoris by commercial corporations requires the user to obtain a commercial license as detailed below. Before using the Pichia Expression Kit, please read the following license a greement. If you do not agree to be bound by its terms, contact Invitrogen within 10 days for authorization to return the unused Pichia Expression Kit and to receive a full credit. If you do agree to the terms of this Agreement, please complete the User Registration Card and return it to Invitrogen before using the kit.INDIVIDUAL PICHIA EXPRESSION KIT LICENSE AGREEMENTInvitrogen Corporation (INVITROGEN) grants you a non-exclusive license to use the enclosed Pichia Expression Kit (EXPRESSION KIT) for academic research or for evaluation purposes only. The EXPRESSION KIT is being transferred to you in furtherance of, and reliance on, such license. You may not use the EXPRESSION KIT, or the materials contained therein, for any commercial purpose without a license for such purpose from RESEARCH CORPORATION TECHNOLOGIES, INC., Tucson, Arizona. Commercial purposes include the use in or sale of expressed proteins as a commercial product, or use to facilitate or advance research or development of a commercial product. Commercial entities may conduct their evaluation for one year at which time this license automatically terminates. Commercial entities will be contacted by Research Corporation Technologies during the evaluation period regarding the purchase of a commercial license.Access to the EXPRESSION KIT must be limited solely to those officers, employees and students of your institution who need access thereto in order to perform the above-described research or evaluation. You must inform each of such officer, employee and student of the provisions of this Agreement and require them to agree, in writing, to be bound by the provisions of this Agreement. You may not distribute the EXPRESSION KIT to others, even those within your own institution. You may transfer modified, altered or original material from the EXPRESSION KIT to a third party following notification of INVITROGEN such that the recipient can be licensed. You may not assign, sub-license, rent lease or otherwise transfer this License or any of the rights or obligation hereunder, except as expressly permitted.This License is effective until terminated. You may terminate it at any time by destroying all Pichia expression products in your control. It will also terminate automatically if you fail to comply with the terms and conditions of the Agreement. You shall, upon termination of the License, destroy all Pichia Expression Kits in your control, and so notify INVITROGEN in writing.This License Shall be governed in its interpretation and enforcement by the laws of the State of California.Product User Registration CardPlease complete and return the enclosed Product User Registration Card for each Pichia Expression Kit that you purchase. This will serve as a record of your purchase and registration and will allow Invitrogen to provide you with technical support and manual updates. It will also allow Invitrogen to update you on future developments of and improvements to the Pichia Expression Kit. The agreement outlined above becomes effective upon our receipt of your User Registration Card or 10 days following the sale of the Pichia Expression Kit to you. Use of the kit at any time results in immediate obligation to the terms and conditions stated in this Agreement.Technical ServicesInvitrogen provides Technical Services to all of our registered Pichia Expression Kit users. Please contact us if you need assistance with the Pichia Expression Kit.Corporate Headquarters: Japanese Headquarters European Headquarters:Invitrogen Corporation1600 Faraday AvenueCarlsbad, CA 92008 USATel: 1 760 603 7200Tel (Toll Free): 1 800 955 6288 Fax: 1 760 602 6500E-mail:tech_service@ Invitrogen Japan K.K.Nihonbashi Hama-Cho Park Bldg. 4F2-35-4, Hama-Cho, NihonbashiTel: 81 3 3663 7972Fax: 81 3 3663 8242E-mail: jpinfo@Invitrogen Ltd3 Fountain DriveInchinnan Business ParkPaisley PA4 9RF, UKTel: +44 (0) 141 814 6100Fax: +44 (0) 141 814 6287E-mail: eurotech@iiiivTable of ContentsTable of Contents (v)Materials (vii)Introduction (1)Overview (1)Vectors (4)pPIC3.5K (5)pAO815 (6)Methods (7)Cloning into pPIC3.5K and pAO815 (7)Analysis of E. coli Transformants (11)pAO815–In Vitro Multimerization Protocol (12)Transformation into Pichia (22)pPIC3.5K–In Vivo Screening of Multiple Inserts (26)Appendix (30)Recipes (30)Pichia Genomic DNA Isolation (31)Easy-DNA™ Protocol for Isolation of DNA from Pichia (33)Determination of Copy Number of Multiple Integrants (34)Technical Service (36)Purchaser Notification (38)References (40)vviMaterialsContents This manual is included with the following catalog numbers:Item AmountCatalogno.pPIC3.5K 20 µg, lyophilized V173-20pAO815 20 µg, lyophilized V180-20Shipping/Storage Lyophilized plasmids are shipped at room temperature and should be stored at -20°C.Materials Supplied by the User For the procedures described in this manual, you will need:• Manual from the Pichia Expression System• Microbiological equipment• Electrocompetent or chemically competent E. coli (must be rec A, end A) for transformation. You will need 3-4 tubes of competent cells per experiment. For protocols to prepare competent E. coli and transformation protocols, please see Current Protocols (Ausubel, et al., 1990) or Molecular Biology: A Laboratory Manual (Sambrook, et al., 1989)• Eco R I, Bam H I and Bgl II restriction enzymes and appropriate buffers• Agarose and low-melt agarose• S.N.A.P.™ Gel Purification Kit (Catalog no. K1999-25) or glass milk• Sterile water• CIP (calf intestinal phosphatase, 1 unit/µl)• 10X CIP Buffer• Phenol/chloroform• 3M sodium acetate• 100% ethanol• 80% ethanol• T4 Ligase (2.5 units/µl)• 10X Ligation Buffer (with ATP)• LB medium• LB-ampicillin plates (50-100 µg/ml ampicillin)• 16°C, 37°C, and 65°C water baths or temperature blocks• Geneticin® antibiotic (Invitrogen, Catalog no. 11811-023)• YPD-Geneticin® plates (see Recipes, page 31)• 50 ml conical centrifuge tubes• Hemacytometer• 30°C and 37°C incubator• Microtiter plates (optional)Important Registered Pichia users should already have the Pichia Expression System and thecurrent manual. Procedures for transformation into E. coli and Pichia, analysis of recombinants, and expression are described in the Pichia manual.continued on next pageviiMaterials, continuedOther PichiaProductsOther Pichia products available from Invitrogen are described below:Item PurposeReactionsorAmountCatalog no.Pichia Expression Kit Complete Kit for GeneExpression in Pichia pastoris10-50 K1701-01Pichia Spheroplast Module Preparation of Pichiaspheroplasts10-50 K1720-01EasyComp™ Kit Rapid preparation andtransformation of competentP. pastoris cells1 kit K1730-015´ and 3´ AOX1 Primers PCR to confirm Pichiarecombinants2 µg each N740-02pPICZ A, B, & C For simple selection onZeocin™ and intracellularexpression of recombinantproteins containing a C-terminal histidine tag20 µg each V190-20pPICZα A, B, &C For simple selection onZeocin™ and secretedexpression of recombinantproteins containing a C-terminal histidine tag20 µg each V195-20pPIC9K Forin vivo isolation of multiplecopy inserts for secretedexpression20 µg V175-20viii1IntroductionOverview Introduction Multiple copy integration of recombinant genes in Pichia has been demonstrated toincrease expression of the desired gene in some cases (Brierley, et al., 1994; Clare, et al., 1991a; Cregg, et al., 1993; Romanos, et al., 1991; Scorer, et al., 1993; Scorer, et al., 1994; Thill, et al., 1990; Vedvick, et al., 1991). The two vectors included in this kit allow isolation and generation of multicopy inserts, either by in vivo or in vitro methods, in order to test whether increasing the copy number of your recombinant gene will lead to a subsequent increase in protein expression. The in vivo method utilizes hyper-resistance to Geneticin ® (G418 sulfate) to screen for possible multicopy inserts, while the in vitro method produces tandem inserts of your gene by ligation.Frequency ofMulticopy Inserts Multiple plasmid integration events occur spontaneously in Pichia at a frequency between 1 and 10% of all His + transformants. The in vivo method allows you to screenfor the His + transformants that may have multiple inserts of your gene. The in vitro method allows you to construct multimers by ligation. When His + transformants are selected, they will have a high probability of containing the multimers that you constructed in vitro .Generation of Multicopy Inserts in vivo pPIC3.5K contains the bacterial kanamycin gene (kan from Tn 903) that confers resistance to Geneticin ® in Pichia . Note that kan does not confer resistance to kanamycin in Pichia . The level of Geneticin ® resistance roughly depends on the number of kanamycin genes integrated. A single copy of pPIC3.5K integrated into the Pichia genome confers resistance to Geneticin ® to a level of ~0.25 mg/ml. Multiple integrated copies of pPIC3.5K can increase the Geneticin ® resistance level from 0.5 mg/ml (1-2 copies) up to 4 mg/ml (7-12 copies). Because of the genetic linkage between the kanamycin gene and the "expression cassette" (P AOX1 and your gene of interest), one can infer that Geneticin ® resistant clones contain multiple copies of your gene. Protein expression may increase because of a gene dosage effect. Thus, the presence of the kan gene on pPIC3.5K can be used as a tool to detect pPIC3.5K transformants that harbor multiple copies of the your gene. The graphic below show multiple insertion and linkage of the kan gene to your expression cassette.(2nd Insertion Event3rd Insertion Event, etc.((continued on next page2 Overview, continuedScreening onGeneticin ® Direct selection of Geneticin ® resistance in yeast does not work well because newly transformed cells need time to express sufficient amounts of the resistance factor. Sinceyeast grow much more slowly than bacteria, significant numbers of recombinant yeast are killed before they accumulate enough of the resistance factor to survive direct plating on antibiotic. Do not use Geneticin ® resistance as a selectable marker. The procedure to generate Geneticin ® resistant clones requires an initial selection of His + transformants followed by a screen for varying levels of Geneticin ® resistance. Resistance toGeneticin ® conferred by the kanamycin gene present on pPIC3.5K is used as a SCREEN, not as a SELECTION for multicopy integrants. Generation of Multicopy Inserts in vitroThe graphic below shows how pAO815 is used to generate multiple expression cassette copies in a single vector prior to transformation into Pichia . The gene of interest is inserted into the vector at a unique Eco R I site. The resulting expression cassette (the P AOX1 plus your gene) is flanked on the upstream side by a unique Bgl II site and on the downstream site by a unique Bam H I site (see A below). The vector containing the gene of interest is digested with Bgl II and Bam H I to excise the expression cassette. The cassette is then reinserted at the Bam H I site to create a tandem repeat of the cassette. The reinsertion process can be repeated to generate a series ofvectors that contain an increasing number of cassettes linked to a single HIS4 gene (see B below).Transformation of Pichia with these in vitro -formed multimers increases the frequency of multicopy expression cassette recombinants. Pichia recombinants may be custom-designed to contain a defined number of multicopy inserts. For more information, please see page 14.RecombinantVectorEco R I Bgl II TT Gene of Interest 5' AOX1P AOX1Eco R I Bam H IInsert Bgl II TT Gene of Interest 5' AOX1P AOX1Bam H I/Bgl II TT Gene of Interest 5' AOX1P AOX1Bam H IVector Digestionwith Bam H I1 Expression Cassette HIS4HIS41 Expression CassetteBam H IHIS4Recombinant Vector2 Expression Cassettes A.B.continued on next pageOverview, continuedWe recommend trying both methods to generate or isolate multicopy inserts of yourgene. A summary of the advantages and disadvantages of each method is presented in thelists below. The "best" method is the one that works for your protein; unfortunately, thereis no way to predict beforehand which method will work for your protein.In vitro Method (pAO815)Advantages Disadvantages • Quantitative--construction of adefined number of multimers• More work up front to clone definednumber of multimers• Most of the His+ transformants willcontain the proper, defined number ofinserts• Size of the vector may become quitelarge depending on the size of yourgene and the number of copies youcreate• Isolation of recombinants withmultiple inserts is easier because mostof the His+ transformants will containmultiple copies of your gene• RearrangementsinE. coli may occur• In vitro construction allows step-wiseanalysis of copy number effects onprotein expression• Multiple inserts are located at a singlelocus• No need for a second drug resistancemarker in the vectorIn vivo Method (pPIC3.5K)Advantages Disadvantages • Easier to initiate experiment becauseonly one copy of your gene is clonedinto pPIC3.5K before transforminginto Pichia• Qualitativescreen--Geneticin®resistance may not necessarilycorrelate with the number of copies ofyour gene.• Identifies the 1-10% of spontaneousHis+ transformants that have multipleinserts• ScreeningHis+ transformants mayinvolve more work because you willneed thousands of His+ transformantsto generate enough Geneticin®resistant colonies to test• Average size of vector is similar toother Pichia expression vectors• The number of multiple inserts isunknown (although this can bedetermined through Southern or dotblot analysis)• Multiple inserts are located at a singlelocus• Screening on Geneticin® is sensitiveto the density of the cells and mayresult in the isolation of false positivesVectorsIntroduction The vectors pPIC3.5K and pAO815 share many of the same features (see below). Both are functional in Pichia strains GS115 and KM71. However, pPIC3.5K has a moreextensive multiple cloning site and contains the kanamycin gene for in vivo screening ofmultiple copy inserts. It is identical to pPIC3.5 except for the presence of the kanamycingene. pAO815 is similar to pHIL-D2 except that it does not contain an f1 origin.Features The table below describes the general features of the pPIC3.5K and pAO815 Pichiaexpression vectors.Feature Description Benefit5´ AOX1An ~1000 bp fragment containing the AOX1 promoter Allows methanol-inducible high level expression in Pichia Targets plasmid integration to the AOX1locus.MCS Multiple Cloning Site Allows insertion of your geneinto the expression vectorTT Native transcription termination andpolyadenylation signal from AOX1 gene(~260 bp)Permits efficient transcriptiontermination and polyadenylationof the mRNAHIS4Pichia wild-type gene coding forhistidinol dehydrogenase (~2.4 kb) andused to complement Pichia his4 strainsProvides a selectable marker toisolate Pichia recombinant strains3´ AOX1Sequences from the AOX1 gene that arefurther 3´ to the TT sequences (~650 bp)Targets plasmid integration at theAOX1 geneAmppBR322originAmpicillin resistance geneE. coli origin of replicationAllows selection, replication, andmaintenance in E. coliBam H IBgl IINot ISac ISal IStu IUnique restriction sites(Note: Stu I is not unique to pPIC3.5K)Permits linearization of vector forefficient integration into thePichia genome and generation ofeither Mut+ or Mut S recombinantskan Kanamycin resistance gene from Tn903which confers resistance to Geneticin®in Pichia and kanamycin resistance in E.coli(for pPIC3.5K only)Allows in vivo screening formulticopy inserts by increasedresistance to Geneticin®Also allows selection forkanamycin resistance in E. coliThere is no yeast origin of replication in any of the Pichia expression vectors available from Invitrogen. His+ transformants can only be isolated if recombination occurs between the plasmid and the Pichia genome (i. e. integration of the plasmid).pPIC3.5KDescription pPIC3.5K is a plasmid designed to allow you to identify in vivo multiple integrations ofyour gene in the Pichia genome. Other details about pPIC3.5K are provided below:• 9004 bp vector• Five unique restriction sites in the multiple cloning site: Bam H I, Sna B I, Eco R I,Avr II, Not I• Intracellular expression of your gene• Requires an initiating ATG codon in a Kozak consensus sequence for proper transla-tion initiation of your gene (Cavener and Stuart, 1991; Kozak, 1987; Kozak, 1990)• HIS4 selection in Pichia• For insertion at AOX1 in GS115 or KM71, linearize with Sac I (generates His+ Mut+in GS115 and His+ Mut S in KM71)• For insertion at HIS4, linearize with Sal I (generates His+ Mut+ in GS115 and His+Mut S in KM71)• For a gene replacement at AOX1 in GS115, linearize with Bgl II (generates His+Mut S)Please see page 23 for alternate restriction sites if your insert DNA has a Bgl II, Sac I, orSal I site.Map of pPIC3.5K The figure below shows the map of pPIC3.5K. Details of the multiple cloning site are shown on page 9.Comments for pPIC3K:9004 nucleotides5´ AOX1 promoter fragment: bases 1-9375´ AOX1 primer site: bases 855-875 Multiple Cloning Site: bases 938-9683´ AOX1 primer site: bases 1055-10753´ AOX1 transcriptiontermination (TT): bases 981-1314HIS4 ORF: bases 4242-1708Kanamycin resistance gene: bases 5458-4656 3´ AOX1 fragment: bases 5850-6607pBR322 origin: bases 7689-7016 Ampicillin resistance gene: bases 8694-7834mHIaBIoRIrIItISal IpAO815Description pAO815 is a plasmid designed for in vitro generation of multimers of your gene forintegration into the Pichia genome. Other details about pAO815 are provided below:• 7709 bp vector• One unique restriction site: Eco R I• Intracellular expression of your gene• Requires an initiating ATG codon in a Kozak consensus sequence for proper transla-tion initiation of your gene (Cavener and Stuart, 1991; Kozak, 1987; Kozak, 1990)• HIS4 selection in Pichia• For insertion at HIS4, linearize with Sal I or Stu I (generates His+ Mut+ in GS115 andHis+ Mut S in KM71)• For a gene replacement at AOX1 in GS115, linearize with Bgl II (generates His+Mut S)Please see page 23 for alternate restriction sites if your insert DNA has a Bgl II, Stu I, orSal I site.Map of pAO815 The figure below shows the map of pAO815. Details of the multiple cloning site are shown on page 10.Comments for pAO815:7709 nucleotides5´ AOX1 promoter fragment: bases 1-940 5´ AOX1 primer site: bases 855-875 Eco R I Site: bases 943-9483´ AOX1 primer site: bases 1024-10443´ AOX1 transcriptiontermination (TT): bases 950-1277 HIS4 ORF: bases 4199-16653´ AOX1 fragment: bases 4554-5310 pBR322 origin: bases 6394-5740oRISal IStu IMethodsCloning into pPIC3.5K and pAO815Introduction We recommend that you ligate your insert into both pPIC3.5K and pAO815 so that you can try both methods to isolate multiple integrants. Below are some guidelines toconsider when developing a cloning strategy for these vectors. The multiple cloning sitesfor each vector are presented on the following pages for your convenience.We recommend that you transform the two supercoiled Pichia expression vectors intoE. coli, so that you have a permanent stock and a way to make more plasmid.• Dilute 1 µl of each plasmid (1 µg/µl) to 10-100 pg/µl using sterile water or TE buffer.• Transform competent E. coli with 1-2 µl of the diluted plasmid and select on LB with50-100 µg/ ml ampicillin (LB-Amp).GeneralConsiderationsThe following are some general considerations applicable to both vectors.• The codon usage in Pichia is believed to be the same as Saccharomyces cerevisiae• Many Saccharomyces genes have proven to be cross-functional in Pichia• Plasmid constructions should be maintained in a rec A, end A mutant E. coli strain suchas TOP10. Electrocompetent TOP10 cells are available from Invitrogen.Item AmountCatalogno.TOP10 Electrocomp™ 5 x 80 µl (400 µl total) C664-55TOP10 Electrocomp™10 x 80µl (800 µl total) C664-11• The native 5´ end of the AOX1 mRNA is noted in each multiple cloning site. This isneeded to calculate the size of the expressed mRNA of the gene of interest if you needto analyze mRNA for any reason.• Translation termination is determined by either stop codons in the gene of interest or inthe 3´ AOX1 sequence. The stop codons in the 3´ AOX1 sequence are noted in eachfigure on the following pages.• The premature termination of transcripts because of "AT rich regions" has beenobserved in Pichia and other eukaryotic systems (Henikoff and Cohen, 1984; Irniger,et al., 1991; Scorer, et al., 1993; Zaret and Sherman, 1984). If you have problemsexpressing your gene, check for premature termination and AT rich regions. It may benecessary to change the sequence in order to express your gene (Scorer, et al., 1993).continued on next pageGeneral Cloning StrategiesStrategies generally fall into three different categories: 1.Ligation of a compatible restriction fragment: a) Forced (directional) insertion involving the use of two different sites in the multiple cloning site (pPIC3.5K).b)Ligation of the fragment with the same restriction end on both ends into a single, compatible site (e.g. Eco R I cloning in pAO815). Note that you will need to dephosphorylate pAO815 to ligate into the Eco R I site.2. PCR amplification of the fragment containing the gene of interest in such a way that compatible restriction ends are generated for ligation into the appropriate vector.3.Direct cloning of an amplified fragment containing the gene of interest via the TA Cloning ®Kit (Catalog no. K2000-01), followed by subcloning of a compatible fragment into the appropriate Pichia expression vector.ImportantIf your insert has an Eco R I site and you are trying to clone into the Eco R I site of pAO815, we recommend the following:1.An enzyme like Bsa I has the following restriction recognition site: 5´-GGTCTCNˇ3´-CCAGAGNNNNNˆ2. An Eco R I site may be engineered into the recognition site for Bsa I.5´-GGTCTCGˇAATTC.....3´-CCAGAGCTTAAˆG.....3. This sequence may be added to your DNA fragment by integrating it into your PCRprimer or created in vitro as an adaptor to another restriction site. 4.Digest your PCR or adapted ligation product with Bsa I. This will generate Eco R I overhangs on both ends of your fragment without digesting with Eco R I. 5´-AATTC......3´-G......5. Ligate into dephosphorylated pAO815. Other enzymes that may be used are Bsm A I or Bsm B I.Cloning ProceduresPlease refer to (Ausubel, et al ., 1990), pages 3.16.1 to 3.17.3. or (Sambrook, et al ., 1989), pages 5.10 to 5.13. for help with cloning.BacterialTransformationOnce you have decided on a cloning strategy, you will need to prepare competent E. coli cells for transformation before setting up your ligation reactions. Please see Current Protocols in Molecular Biology (Ausubel, et al ., 1990) or Molecular Biology: A Laboratory Manual (Sambrook, et al ., 1989) for preparation of electrocompetent or chemically competent E. coli or use your laboratory's procedure.continued on next pageP AOX1 and Multiple Cloning Site ofpPIC3.5K The sequence below shows the details of the multiple cloning site and surroundingsequences.ATATAGTATA GGATTTTTTT TGTCATTTTG TTTCTTC AOX1 mRNA 3´ end (1146)SpecialConsiderations• For pPIC3.5K, the fragment containing the gene of interest should have a Kozakconsensus sequence for proper translation initiation, although this requirement is not as stringent in yeast. For example, ACC ATG G is a Kozak consensus sequence, where the ATG corresponds to the initiating ATG for your gene of interest (Cavener and Stuart, 1991; Kozak, 1987; and Kozak, 1990). Note : There is an ATG upstream of the Sna B I site.• Be sure to analyze the 5´ untranslated region of the mRNA for secondary structure formation. Secondary structure in the mRNA may have a negative effect on expression of the recombinant protein.• If you are digesting with Bam H I and Sna B I or Sna B I and Eco R I, digest with Sna B I first. If you digest with Bam H I or Eco R I first, the Sna B I site will be too close to the end of the DNA and will not be digested properly.continued on next pageP AOX1 and Multiple Cloning Site of pAO815 The sequence below shows the details of the multiple cloning site and surrounding sequences.TTATCATCAT TATTAGCTTA CTTTCATAAT TGCGACTGGT TCCAATTGAC AAGCTTTTGA TTTTAACGAC TTTTAACGAC AACTTGAGAA GATCAAAAAA CAACTAATTA TTCGAAACGA GGAATTCGCC TTAGACATGA CTGTTCCTCA GTTCAAGTTG GGCACTTACG AGAAGACCGG TCTTGCTAGA TTCTAATCAA GAGGATGTCA GAATGCCATT TGCCTGAGAG ATGCAGGCTT CATTTTTGAT AOX1 mRNA 5´ end (824)Eco R I5´AOX 1 primer site (855-875)3´AOX 1 primer site (1024-1044)AOX1 mRNA 3´ end (1115)Special Considerations • For in vitro multimerization, you need to analyze your insert for Bam H I and Bgl II restriction sites. If your insert has a Bam H I or Bgl II site, we recommend that you use the in vivo method (pPIC3.5K) to isolate multiple inserts of your gene.• For pAO815, the fragment containing the gene of interest should have a Kozak consensus sequence for proper translation initiation, although this requirement is not as stringent in yeast. For example, ACC ATG G is a Kozak consensus sequence, where the ATG corresponds to the initiating ATG for your gene of interest (Cavener and Stuart, 1991; Kozak, 1987; and Kozak, 1990).• Be sure to analyze the 5´ untranslated region of the mRNA for secondary structure formation. Secondary structure in the mRNA has a negative effect on expression of the recombinant protein.。

西安大学生物工程学院2020级《生物化学》考试试卷(3885)

西安大学生物工程学院2020级《生物化学》课程试卷（含答案）__________学年第___学期考试类型：（闭卷）考试考试时间：90 分钟年级专业_____________学号_____________ 姓名_____________1、判断题（50分，每题5分）1. HIV蛋白酶与胃蛋白酶一个主要的差别是后者的活性中心位于一个单一的亚基上，而前者的活性中心由两个亚基参与构建。

（）答案：正确解析：2. 天然存在的不饱和游离脂肪酸大多具有反式结构。

（）[山东大学2017研]答案：错误解析：天然存在的不饱和游离脂肪酸大多具有顺式结构，少部分具有反式结构。

3. 功能蛋白质分子中，只要有氨基酸残基发生改变都会引起生物功能的丧失。

（）[武汉科技大学2018研]答案：错误解析：一个典型蛋白质的功能决定于它三维结构，而蛋白质所采取的三维结构又决定于它的氨基酸序列。

若由于氨基酸残基发生的改变影响了该蛋白质的三维结构，则会哺乳动物引起其生物功能的丧失，但若未影响其三维整体，则不一定会引起生物功能的丧失。

4. 酶的pH对酶活性的曲线均为钟罩形。

（）。

答案：错误解析：虽然大多数酶的pH～酶活性曲线为钟罩形，但并不是所有的酶都如此，有的只有钟罩形的一半，如胃蛋白酶、胆碱酯酶；有的甚至是直线，如木瓜蛋白酶。

5. 原核mRNA中的修饰碱基比真核mRNA的多。

（）答案：错误解析：原核mRNA与真核mRNA相比5′端无帽状结构存在，3′端不含polyA结构。

6. 在多肽分子中只存在一种共价键即肽键。

（）答案：错误解析：在多肽分子中除肽键外，还有二硫键等其他共价键。

7. 神经酰胺也是一种第二信使。

（）答案：正确解析：8. 真核生物成熟mRNA的两端均带有游离的3′OH。

（）答案：正确解析：真核生物成熟mRNA的5′为帽子结构即，因此5′端也是3′OH。

9. 蛋白质变性和DNA变性的机理是相似的，复性时都可以恢复原来的生物活性。

（）答案：错误解析：10. 在酶的纯化实验过程中，通常会丢失一些活性，但偶尔亦可能在某一纯化步骤中酶的活力可超过100，可能是由于此酶的激活剂的加入或抑制剂的丢失造成的。

苹果提取物对喉癌细胞增殖的抑制作用

研究背景
·流行病学研究发现苹果的消费与降低心血管疾病以
及某些癌症的危险性有关。
· 美国康奈尔大学研究所,《Fresh appleinhibits
breast tumor cells in rats, the proliferation of
inducing apoptosis its generation》
苹果提取物的制备和储存
技术路线
喉癌细胞的复苏与培养
实验分组（10个不同组）
MTT法测定对喉癌细胞增殖抑制作用
倒置显微镜观察细胞形态 Hochest染色荧光显微镜观癌细胞核形态变化 PI染色,流式细胞术分析细胞周期记录结果分析讨论
关键问题及可行性
• 苹果提取物制备、最佳抑制浓度的确定 • 旋转蒸发仪、荧光倒置显
· 郑州大学教授苹果提取物对食管癌细胞
增殖、凋亡及相关蛋白表达的影响Βιβλιοθήκη 人类研究抗喉癌有了新的思路：
苹果提取物
苹
果
提
取
物
·通过丙酮法萃取苹果，由于含有较多酚类物
质，酪酸，丰富的果胶，维生素c等,具有抗氧化和肿瘤化学预防的作用，并可抑制某些肿瘤细胞生长。
目的与意义
· 苹果提取物对喉癌HEP-2细胞株增殖、凋亡的影响,
微镜、流式细胞仪、酶联
免疫检测仪等各仪器的联
系和使用
• 试剂提供与购买
• 时间的调配和经费预算
谢谢
苹
•
• • • •
果
提
取物制备
将新鲜苹果洗净去皮去籽后均匀切成2mm 薄片放入带盖的烧瓶中并置于冰块上, 以减少由于酶化反应而引起的颜色褐变。 100g苹果中加入 200ml 80% 丙酮( 1：2)在匀浆器内搅拌2min。取出匀浆液放入接有真空泵的滤过瓶中,放置定性滤纸,抽滤。收集滤过物置于旋转蒸发器中在45度条件下旋转蒸发45min。样品用去离子水调节到终体积为 100ml并于- 80 度储存。

DL-苹果酸食品添加物英文公定书

solution does not disappear within 3 minutes. Residue on Ignition Not more than 0.05% (5 g). Assay Weigh accurately about 1.5 g of DL-Malic Acid, and dissolve it in water to make exactly 250 ml. Measure exactly 25 ml of this solution, and titrate with 0.1 mol/L sodium hydroxide (indicator: 2 drops of phenolphthalein TS). Each ml of 0.1 mol/L sodium hydroxide = 6.704 mg of C4H6O5
C6H6O3 Mol. Wt. 126.11 3-Hydroxy-2-methyl-4H-pyran-4-one [118-71-8] Content Maltol contains not less than 99.0% of maltol (C6H6O3). Description Maltol occurs as white or slightly yellowish needles or crystalline powder having a sweet odor. Identification Determine the absorption spectrum of Maltol as directed in the Paste Method under Infrared Spectrophotometry, and compare with the Reference Spectrum of Maltol. Both spectra exhibit similar intensities of absorption at the same wavenumbers. Purity (1) Melting point 160–163°C. (2) Clarity of solution Clear (0.10 g, 70% (vol) ethanol 5.0 ml). (3) Heavy metals Not more than 10 µg/g as Pb (2.0 g, Method 2, Control solution Lead Standard Solution 2.0 ml). (4) Arsenic Not more than 4.0 µg/g as As2O3 (0.50 g, Method 4, Apparatus B). Loss on Drying Not more than 0.5% (4 hours). Residue on Ignition Not more than 0.05%. Assay Test Solution Weigh accurately about 0.2 g of Maltol, and dissolve it in 0.1 mol/L hydrochloric acid to make exactly 500 ml. Measure exactly 5 ml of this solution, and add 0.1 mol/L hydrochloric acid to make exactly 200 ml. Standard Solution Weigh accurately about 0.2 g of maltol for assay, and dissolve it in 0.1 mol/L hydrochloric acid to make exactly 500 ml. Measure exactly 5 ml of this solution, and add 0.1 mol/L hydrochloric acid to make exactly 200 ml. Procedure Measure the absorbances (AT and AS) of the test solution and the standard solution at a wavelength of 274 nm, using 0.1 mol/L hydrochloric acid as the reference. Calculate the content by the formula:

苹果的一段作文英语

苹果的一段作文英语Title: The Enigmatic Apple: A Fruit of Global Significance。

Introduction:The apple, a fruit of multifaceted symbolism and practical utility, has entrenched itself deeply into human culture and history. From its origins in Central Asia to its global dissemination, the apple has traversed borders, languages, and imaginations, leaving an indelible mark on societies worldwide.Historical Significance:Dating back to ancient times, the apple holds a significant place in mythology and folklore. In Greek mythology, the golden apple sparked the Trojan War, symbolizing discord and rivalry. Conversely, in Norse mythology, apples represented eternal youth and vitality,consumed by the gods to retain their immortality. Such divergent interpretations highlight the fruit's enigmatic nature and its ability to embody various meanings across different cultures.Cultural Symbolism:Beyond mythology, the apple has permeated cultural expressions, appearing in art, literature, and religious texts. In the biblical narrative of Adam and Eve, the forbidden fruit is often depicted as an apple, signifying temptation and the loss of innocence. Moreover, artists like Cézanne and Van Gogh immortalized the apple in their paintings, capturing its form and color in timeless works of art. In literature, from Shakespeare's "Romeo and Juliet" to Robert Frost's "After Apple-Picking," the fruit serves as a metaphor for love, desire, and the passage of time.Nutritional Value and Health Benefits:From a nutritional standpoint, the apple offers amyriad of health benefits. Rich in dietary fiber, vitamins, and antioxidants, it promotes digestive health, boosts immunity, and reduces the risk of chronic diseases. Moreover, its low calorie and high water content make it an ideal snack for weight management and hydration. Whether consumed fresh, juiced, or dried, the apple serves as a wholesome addition to a balanced diet, earning its reputation as a "miracle fruit."Economic and Agricultural Importance:Beyond its cultural and nutritional significance, the apple holds immense economic and agricultural value. Cultivated in diverse climates and regions worldwide, apples contribute significantly to global agricultural production and trade. Leading apple-producing countries such as China, the United States, and Poland drive the industry's growth, supplying fresh apples, juices, and processed products to international markets. Furthermore, the cultivation of apples supports livelihoods in rural communities, providing employment opportunities and economic stability.Environmental Impact and Sustainability:However, the widespread cultivation of apples also poses environmental challenges, including pesticide use, water consumption, and soil degradation. To mitigate these issues, sustainable farming practices such as integrated pest management, water conservation, and soil conservation are being increasingly adopted. Furthermore, initiatives promoting organic farming and agroecology aim to minimize the environmental footprint of apple production while preserving biodiversity and ecosystem health.Conclusion:In conclusion, the apple stands as a symbol of cultural heritage, nutritional abundance, economic prosperity, and environmental stewardship. Its journey from ancient mythologies to modern supermarkets reflects humanity's enduring fascination with this humble fruit. As we continue to explore its mysteries and harness its benefits, let usalso strive to cultivate and consume apples in a manner that sustains both the planet and future generations.。

苹果基因组芯片 (Axiom_Apple480) 使用说明书

Axiom ® Apple Genotyping Array (Axiom_Apple480) was designed through the Expert Design Program at Affymetrix in collaboration with the FruitBreedomics consortium (). The sequencing and marker selection was conducted by experts from Fondazione Edmund Mach, INRA, Dalhousie University, Wageningen UR, University Di Bologna, and Universita’ Degli Studi Di Milano.Apple (Malus domestica ) is one of the most cultivated plants in the world. The apple genome is an ancient tetraploid, with some varieties being either allotetraploid or triploid. The apple genome is highly polymorphic with approximately 1 single-nucleotide polymorphism (SNP) per 50 bp and has a rapid linkage disequilibrium (LD) decay (20–55 kb). Axiom Apple Genotyping Array includes 480,000 markers and together with Axiom™ Analysis Suite software overcomes the genotyping challenges associated with polyploidy, rapid LD decay, and high polymorphism observed in the apple genome.The 96-format array includes markers identified using whole-genome sequence data from 63 Malus domestica cultivars and two double haploid accessions. T able 1 lists the number of cultivars and corresponding country of origin used in the SNP discovery process. The names of these cultivars are provided in T able 2.Array highlightsnVery high diversitynIncludes markers discovered in 63 worldwide M. domestica cultivars nHigh resolution to address rapid decay of LDn487,249 markers on the arraynBias towards common variants with minor allele frequency (MAF) >0.05nIncludes 21,463 previously validated markers: 19,990 markers from an existing in-market 20K Fruitbreedomics apple array and 1,473 markers identified using genotyping-by-sequencing (GBS)nAbsence of paralogous variants through the use of double haploid accessions in SNP discoveryApplicationsn Construction of high-resolution genetic maps n Fine mapping of quantitative trait loci n Genome-wide association studies nSelection sweep analysis1Axiom ® Apple Genotyping ArrayThe most comprehensive high-density apple genotyping arrayData SheetComprehensive coverage of world-wide diversity in applesThe 63 cultivars used in sequencing represent diverse apple germplasm and include some of the core European apple breeding founder varieties. These cultivars were chosen to maximize the genetic diversity in the SNP discovery phase. Two double haploid (DH) accessions, `X9273’ and `X9748’ derived from Golden Delicious, were included to identify pseudo-SNPs created from the erroneous assembly of paralogous regions of the apple genome.Markers that have been previously validated and associated with desirable traits are very important in maintaining and breeding elite commercial populations. The inclusion of 19,990 markers from the existing in-market 20K Fruitbreedomics genotyping array ensures that the new Axiom® array can be used for comparison with data generated by previous studies. The data analysis with Axiom Analysis Suite overcomes the limitations associated with poor coverage and the challenging analysis1 of genotype data observed in the 20K in-market apple genotyping array. The backwards compatibility also provides the ability to continue existing projects, while making use of the latest and most informative content, to extend the usefulness of the study. The performance of the existing in-market array markers is less than ideal because the markers represent a small set of core founder lines, the arrays have a very low density making it difficult to work with the rapid LD decay in the apple genome, and data analysis software is not suitable for polyploid analysis.A key benefit of the Axiom array is the capability to genotypeSNPs that may have neighboring markers as few as 20 bp away.This design feature is important in genotyping the highlypolymorphic structure of apple. The array manufacturingtechnology from Affymetrix also guarantees 100% fidelity andensures all markers are present on every manufacturing batch,unlike other technologies that experience batch-to-batchvariability and SNP dropouts.Genotyping is performed using Axiom Analysis Suite in aconvenient 96 format. With one-click analysis, hands-on time forgenotyping is reduced, minimizing costs and time to results.Axiom Analysis Suite genotypes and classifies the markers into sixeasy-to-visualize categories. The AxiomGT1 algorithm is the onlyalgorithm that adapts to shifted clusters and cluster compressionthat is typically observed in polyploid species, eliminating theneed for manual editing of the clusters and manual assignmentof genotypes.Array designThe markers on the array were identified from whole-genomedata from 63 cultivars. The average number of reads for eachcultivar was 95.2 million, which represents a mean sequencingdepth of 25X. Sequencing reads were mapped as single ends onthe reference genome.2 A total of 15.5 million markers wasidentified from the whole-genome sequencing data. A putativelist of 12,701,549 markers was submitted to Affymetrix tocalculate in silico design scores. The putative list was generatedby removing (i) markers with a low-quality phred score (<20), ahigh combined read depth (>4,000), and a low single-cultivarread depth (<8) in more than 50% of the sequenced cultivars; (ii)heterozygous markers identified in the DH cultivars because theseare evidence of paralogous sequences; and (iii) insertion ordeletions. The in silico design pipeline developed by Affymetrixidentified the following additional markers that were then alsoremoved: (i) markers with low in silico design score (<0.6), (ii)markers with 16-mer count >300 in the genome, (iii) multi-allelicmarkers, (iv) A/T or C/G transversions, and (v) markers with a SNP35 bases up/downstream.The remaining 2.8 million markers were used for choosingmarkers for array synthesis. The following criteria were applied tochoose all the tag markers within a ±10 kbp window: (i) markers2in genic regions with high MAF ≥0.1 and a Hardy-Weinberg Fisher’s test p-value >10-8 with less than 32 missing genotypes, (ii) markers in intergenic regions with MAF >0.1 and Hardy-Weinberg Fisher’s test p-value >10-8 with less than 14 missing genotypes, and (iii) genomic markers with 0.05 ≤ MAF <0.1 with Hardy-Weinberg Fisher’s test p-value >10-8 with less than 14 missing genotypes. The 465,786 markers identified using this method were then combined with 21,463 previously validated markers from the in-market 20K Fruitbreedomics array and GBS data. The resulting 487,249 markers selected for array synthesis represented 40,192 sequence contigs and 562 Mb of the apple genome.Automated genotyping and classificationAxiom Apple Genotyping Array was evaluated with a diverse set of cultivars to demonstrate the array’s performance. A total of 1,200 samples were processed and analyzed using Axiom Analysis Suite, as per the Axiom ® Genotyping Solution Data Analysis Guide (PN 702961 Rev. 3). Approximately 360,565 or 74% of the markers were automatically identified as high-quality markers under the polymorphic high-resolution (PolyHighResolution) category. The call rate of markers in this category was greater than 99%. The data was automatically clustered, assigned genotypes, and classified into six categories for easy visualization. SNP concordance with sequencing was carried out by genotyping 42 of the 63 accessions used in SNP discovery. A total of 347,805 markers in the PolyHighResolution category had 96% concordance, demonstrating the success of the array and the appropriate selection of the cultivars for SNP discovery.3Table 3: Axiom ® Apple Genotyping Array results assigned into six categories. The third column displays the classification of the markers that are available on the legacy apple array with 20,000 markers. The markers in the recommended categories include: 1) PolyHighResolution markers: markers demonstrating three clusters with good cluster resolution and at least two examples of the minorallele; 2) NoMinorHomozygous markers: markers exhibiting two clusters with no examples of the minor allele; 3) MonoHighResolution markers: markers demonstrating a single cluster; 4) OffT argetVariant markers: reproducible yet uncharacterized variants caused by double deletion, sequence non-homology, or DNA secondary structure.SNP classification Percentage of all markers in the different SNP categories (%)Percentage of markers that were previously validated on 20K in-market array and GBS (%)All markers100%100%Recommended markers nPolyHighResolution 7457 nNoMinorHomozygous 25 nMonoHighResolution 14nOffTargetVariant13Unexpected heterozygosity 29High variance62References1. Bianco L., et al. Development and validation of a 20K single nucleotide polymorphism (SNP) whole genome genotyping array for apple (Malus ×domestica Borkh). PLoS ONE9(10): e110377 (2014).2. Velasco R., et al. The genome of the domesticated apple (Malus domestica Borkh.) Nature Genetics42(10):833{9} (2010).Affymetrix, Inc: (US) +1-888-362-2447, +1-408-731-5000n(EU) +44-(0)1628-552550n(JP) +81-(0)3-6430-4020n(CN) +86-21-63915511 eBioscience Products: (US) +1-888-999-1371, +1-858-642-2058n(EU) +43 1 796 40 40 305n(JP) +81-(0)3-6430-4020USB Products: (US) +1-800-321-9322, +1-216-765-5000n(EU) +44-(0)1628-552600 Please visit our website for international distributor contact information.For Research Use Only. Not for use in diagnostic procedures.P/N GGNO06531 Rev. 1© 2015 Affymetrix, Inc. All rights reserved. Affymetrix®, Axiom®, Command Console®, CytoScan®, DMET™, GeneAtlas®, GeneChip®, GeneChip-compatible™, GeneTitan®, Genotyping Console™, myDesign™, NetAffx®, OncoScan®, Powered by Affymetrix™, PrimeView®, Procarta®, and QuantiGene® are trademarks or registered trademarks of Affymetrix, Inc. All other trademarks are the property of their respective owners.4。

苹果超氧化物歧化酶的提取与纯化

苹果中超氧化物歧化酶的提取与纯化一实验原理1969年Mccord和Fri-dorich首次从牛血中提纯了超氧化物歧化酶(Superoxide Dismutase)[1]。

SOD是广泛存在于各种生物体内中, 人们发现靠氧呼吸的所有生物中都存在SOD，如微生物、植物和动物体[2]。

其含量随生物体的不同而不同，即使同一种生物的不同组织或同一组织的不同部位其SOD的种类和含量也有很大差别。

SOD属于金属酶,根据所含金属不同,分为Cu-Zn SOD, Mn-SOD和Fe-SOD三种,三种酶都可催化O-2歧化为H2O2和O2,但其性质有所不同[3] 。

同时SOD是目前为止所发现的唯一的以O2-为底物的酶,能催化细胞内超氧化物阴离子(O2-)的歧化反应,使O2-转化为H2O2和O2[4]。

从植物中提取SOD已有不少报道,但从苹果中提取SOD尚未见报道，本实验以苹果为原料，探究SOD的提取工艺。

目前植物中提取SOD采用的方法主要包括磷酸缓冲液提取法、Tris-HCl和热变性法,磷酸缓冲液法用的较多，本实验也采用这种方法，但相鉴于一些学者的研究得出热变性法是提取工艺中一种较为廉价的方法,可以降低生产成本,并且简单易行。

热变性法不引入其他杂质,对提取工艺非常有利[5]。

由于SOD是一种热稳定性较好的酶,并且大部分杂蛋白在55℃时就可变性[6]。

因此本实验利用SOD和杂蛋白变性温度的差异来加强初步分离的效果。

粗取得到的SOD粗酶液必须进一步的纯化，离子交换柱层析方法是纯化蛋白质中比较常用的方法，它是依据化合物与离子交换剂的结合力的不同而进行分离纯化的。

离子交换柱层析的固定相是离子交换剂，也液体为流动相。

离子交换剂是一种不溶于水的惰性高分子聚合物基质，通过一定的化学反应共价结合上某种电荷基团形成。

酶活力（enzyme activity）也称为酶活性，是指酶催化一定化学反应的能力。

酶活力的大小可用在一定条件下，酶催化某一化学反应的速度来表示，酶催化反应速度愈大，酶活力愈高，反之活力愈低。

常见重组蛋白纯化方法

常见重组蛋白的纯化韦新桂(Chromatography) 北京韦氏博慧色谱科技有限公司，,weixingui@前言：近年来随着生物技术的进步，特别是基因工程的迅猛发展，表达蛋白已经变得很容易，相对而言，纯化却是一个非常繁杂的工作，所以越来越多的研究者把需要表达的目标蛋白和亲和纯化用的标签融合表达，这样纯化相对得比较容易，即使如此，由于蛋白的多样性，纯化依然是比较复杂而专业的工作，尤其对于不熟悉纯化的研究者而言，它成为一个项目的瓶颈，本手册就是把一些相关的材料汇集，希望对纯化重组蛋白有所帮助。

1．1常见纯化的标签理想的标签需要有以下的几个特点，1最好能一步纯化得到纯品；2对目标蛋白的结构和活性没有影响；3方便切除标签；4 应用范围广，可适用各种表达系统或目标蛋白。

但是没有哪个标签是完美的，只能根据实际需要去自己筛选，下表是部分的标签以及纯化的方案：标签纯化用的填料或配基洗脱方法多聚组氨酸（6XHis）螯合镍、铜、钴离子的填料咪唑或降低pH谷胱甘肽硫转酶（GST）键合谷胱甘肽的亲和填料 10-20mM还原谷胱甘肽麦芽糖结合蛋白（MBP）淀粉琼脂糖凝胶麦芽糖金黄色葡萄球菌蛋白A IgG琼脂糖凝胶低pHFlag peptide 抗Flag抗体 ,M1,M2 低pH或EDTA多聚精氨酸（Poly-Arg） SP琼脂糖凝胶高盐多聚半胱氨酸（Poly-Cys）活化巯基琼脂糖凝胶 DTT多聚苯丙氨酸（Poly-Phe）苯基琼脂糖凝胶乙二醇钙调蛋白结合肽钙调蛋白 EGTA 纤维素结合域纤维素盐酸胍或脲几丁质结合域几丁质巯基乙醇，半胱氨酸由于篇幅有限，手册只只写多聚组氨酸标签、GST融合蛋白。

2．组氨酸标签蛋白的纯化His-Tag融合蛋白是目前最常见的表达方式，而且很成熟，它的优点是表达方便而且基本不影响蛋白的活性，无论是表达的蛋白是可溶性的或者包涵体都可以用固定金属离子亲和色谱去（IMAC）纯化。

2．1 IMAC(Immobilized Metal-ion affinity chromatography)是Porath et al.1975年用固定IDA作为配基的填料螯合过渡金属铜、镍、钴或锌离子，可以吸附纯化表面带组氨酸、色氨酸或半胱氨酸残基的蛋白，1987年Smith et al. 发现带有几个组氨酸或色氨酸小肽和螯合金属离子的IDA-sephadex G-25作用力更强，此前在1986年他和他的合作者用Ni2+-IDA-sephadex G-25亲和纯化在氨基端带组氨酸和色氨酸的胰岛素原。

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Apple Regu35-K
Scope | Definitions | Restricted Substances in Products | Reportable Substances and Future Restrictions in Products | Notifying Apple of Chemical Phase Out and Reformulation from Suppliers | Restrictions in Manufacturing Processes Reportable Substances and Future Restrictions in Manufacturing Processes | Supplementary Specifications | Demonstrating Compliance | Waiver Process | Full Material Disclosure (FMD) | Revision History | Referenced Documents | Appendices
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Effective Date: This specification takes effect on September 1, 2018. Prior to this date, revision J of the Regulated Substances Specification is in effect.
1. Scope
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This Regulated Substances Specification describes Apple’s global restrictions on the use of certain chemical substances or materials in Apple’s products, accessories, manufacturing processes, and packaging used for shipping products to Apple’s end-customers. Restrictions are derived from international laws or directives, regulatory agency or eco-label requirements, and Apple policies. Apple’s restrictions may go beyond regulatory requirements in order to protect human health and the environment.