抑制差减杂交
抑制性减法杂交技术
抑制性减法杂交技术1996年，L. Diatchebko在RDA的基础上建立了抑制性减法杂交（supression subtractive hybridization, SSH）技术，该技术是RDA技术的发展，能有效克服RDA或cDNA RDA技术难以解决的问题，如两者不能用于分离两组基因表达差异较小的基因，也不能用于研究存在上调表达的基因等。
(1) SSH的主要原理SSH的核心技术是抑制性PCR (suppression PCR )，它是一种将检测子cDNA单链标准化步骤和消减杂交步骤结合为一体的技术。
其中标准化步骤均等了检测子中的cDNA单链丰度，而消减杂交步骤去除了检测子和驱赶子之间的共同序列，使检测子和驱赶子之间不同的序列得到扩增。
因此SSH显著增加了获得低丰度表达差异cDNA的概率，简化了对消减文库的分析。
抑制PCR是利用链内复性优先于链间复性的原理，使非目标序列片段两端的长反向重复序列(long inverted repeats)在复性时产生“锅柄样”(panhandle-like）结构或“发夹结构”而无法与引物配对，从而选择性地抑制了非目标序列的扩增。
同时，根据杂交的二级动力学原理，丰度高的单链cDNA复性时产生同源杂交速度要快于丰度低的单链cDNA，从而使得丰度存在差异的cDNA相对含量趋于基本一致。
（2）SSH的基本过程如图所示，SSH的主要步骤包括：限制性核酸内切酶切割，产生大小适当的平头末端cDNA片段。
②将检测cDNA分成均等的两份，分别接上接头1或接头2,接头（adaptor)由一长链(40nt)和一短链(l0nt)组成的一端是平末端的双链cDNA分子。
长链3′端与cDNA5′端相连。
长链外侧序列（约20nt）与第一次PCR引物序列相同，内侧序列则与第二次PCR引物序列相同。
此外，接头上含有升启动子序列及内切酶识别位点（如Not I, Srf I, Sma I和Xba I等），为以后将该片段插入克隆载体和测序提供便利。
抑制性差减杂交技术_SSH_在植物学研究中的应用
抑制性差减杂交技术( SSH) 在植物学研究中的应用
阎爱华, 王冬梅
( 河北农业大学 生命科学学院, 河北 保定 071001)
摘要 : 抑制性差减杂交是一种基于转录水平的杂交技术, 能将差异表达 基因扩增 千倍后富集, 具有 高效、灵 敏、操 作简单, 假阳性率低等特点, 已经越来越多的被应用在植物学研究领域。就该 技术的原理、特点 及其在植物 学方面的 应用研究进展作一简要介绍。
DNA 微阵列技术 DNA microarray
巨克隆技术 M egaclone
将基因片段、寡聚核苷酸、cDNA 等固定 在硅质、塑料、玻 璃或尼龙膜 上, 用不 同组 织或 来源 的 mRNA 制 成探 针, 与芯片杂 交, 根 据信 号 查找 差 异 片段 并 进行 克 隆 和分 析。 cDNA 连上不 同的标签 ( tag ) , 与 带有 antitag 的 microbeads 杂交, 固定 cDNA, 并 且每 个 microbead 上 只与 一个 c。
基因差异表达分析方法
Methods of different gene expression techniques
基本原理
The basic principles of different gene expression techniques
优点 Advantage
缺点 Disadvantage
mRNA 差异显示 PCR mRNA differential display
收稿日期: 2008- 08- 11 基金项目: 国家自然科学基金资助项目( 30671244) ; 河北省应有基础研究计 划重点基础研究 项目资助( 08965505D) ; 河北省自然科 学基金
抑制差减杂交技术原理及常见操作结果分析
将RsaI酶切完全的cDNA用adapter1和adapter2R两 种接头连接。通过PCR扩增检测连接效率。 PCR的引物根据RsaI位点和adapter1、adapter2R 的序列设计。这一般要求扩增的片段中不含RsaI 位点序列。在扩增后琼脂糖胶电泳检测结果中, 如果用一个基因特异性引物和一个接头引物所得 到的带亮度与两个基因特异性引物所得到的带的 亮度一致,说明连接较好;如果用一个基因特异性 引物和一个接头引物所得到的带亮度只有两个基 因特异性引物所得到的带的亮度的25%,则说明连 接效率不到25%,应检查RsaI酶切效果并重新连接。
3 抑制差减杂交技术的优缺点
3.1 与其他几种方法相比,SSH技术具有较明显的优越性: (1)通过两步消减杂交和两次抑制PCR可DDRTPCR和cDNA-RDA法中,低丰度的 mRNA一般不易被检测到,SSH方法所做的均等化和目 标片段的富集,保证了低丰度mRNA 也可被检测出。 (3)速度快,效率高。一次SSH反应可 以同时分离几十或成百个差异表 达基因。
抑制差减杂交技术原理 及常见操作结果分析
摘要
抑制差减杂交 (SuppressionSubtractiveHybridization,SSH)是一种 高效鉴定和分离克隆差异表达基因的新技术。 目前,该技术在分子生物学研究的各个领域得 到了广泛的应用作了较全面的介绍,可为研究者们 提供参考。
2.4 第一次杂交、第二次杂交和PCR扩增
将经RsaI消化的drivercDNA和分别连接有adapter1、 adapter2R的testercDNA混合进行第一次杂交。再将第一次 杂交的两个样本混合在一起,同时加入新鲜的drivercDNA, 以进一步富集差异表达序列。在两端具不同接头的差异 表达的cDNA形成新的杂交分子。
抑制性差减杂交技术及其在柑橘上的应用
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杨 明，等 ：抑制性差 减杂交技术及其在柑橘上的应用
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摘要 ：抑制性差减杂交技术是 一种 高效 分离和鉴定差异表达基 因的生物学手段 。介 绍了抑制性差减杂 交技术 的基本 原理、技术方法 以及在柑橘 中的应用 。 关键词 ：抑制性差减杂交 ；差 异表达基 因；柑橘
分子生物学常用实验方法原理介绍
分子生物学常用实验方法原理介绍一、GST pull-down实验基本原理:将靶蛋白-GST融合蛋白亲和固化在谷胱甘肽亲和树脂上,作为与目的蛋白亲和的支撑物,充当一种“诱饵蛋白”,目的蛋白溶液过柱,可从中捕获与之相互作用的“捕获蛋白”(目的蛋白),洗脱结合物后通过SDS-PAGE电泳分析,从而证实两种蛋白间的相互作用或筛选相应的目的蛋白,“诱饵蛋白”和“捕获蛋白”均可通过细胞裂解物、纯化的蛋白、表达系统以及体外转录翻译系统等方法获得。
此方法简单易行,操作方便。
注：GST即谷胱甘肽巯基转移酶(glutathione S-transferase)二、足印法（Footprinting）足印法（Footprinting）是一种用来测定DNA-蛋白质专一性结合的方法，用于检测目的DNA序列与特定蛋白质的结合，也可展示蛋白质因子同特定DNA片段之间的结合。
其原理为：DNA和蛋白质结合后，DNA与蛋白的结合区域不能被DNase（脱氧核糖核酸酶）分解，在对目的DNA 序列进行检测时便出现了一段无DNA序列的空白区(即蛋白质结合区)，从而了解与蛋白质结合部位的核苷酸数目及其核苷酸序列。
三、染色质免疫共沉淀技术（Chromatin Immunoprecipitation，ChIP）染色质免疫共沉淀技术（Chromatin Immunoprecipitation，ChIP）是研究体内蛋白质与DNA相互作用的有力工具，利用该技术不仅可以检测体内反式因子与DNA的动态作用，还可以用来研究组蛋白的各种共价修饰以及转录因子与基因表达的关系。
染色质免疫沉淀技术的原理是：在生理状态下把细胞内的DNA与蛋白质交联在一起，通过超声或酶处理将染色质切为小片段后，利用抗原抗体的特异性识别反应，将与目的蛋白相结合的DNA 片段沉淀下来。
染色质免疫沉淀技术一般包括细胞固定，染色质断裂，染色质免疫沉淀，交联反应的逆转，DNA的纯化及鉴定。
四、基因芯片（又称 DNA 芯片、生物芯片）技术基因芯片指将大量探针分子固定于支持物上后与标记的样品分子进行杂交，通过检测每个探针分子的杂交信号强度进而获取样品分子的数量和序列信息。
抑制性差减杂交
BIOLOGIA PLANTARUM 52 (3): 486-492, 2008486Suppression subtractive hybridization identifies differentially expressed genes in Brassica napus chlorophyll-reduced mutantH.Y. WANG, Y.H. HU, Y. LIU, Y.T. ZHOU, M.L. WANG and Y. ZHAO*Key Laboratory of Ministry of Education for Bio-resources and Eco-environment, College of Life Science, Sichuan University, Chengdu 610064, P.R. ChinaAbstractSuppressive subtraction hybridization (SSH) was used to identify differentially expressed genes caused by a chlorophyll-reduced mutation in B. napus . The cDNA fragments, derived from SSH positive subtractive library (tester: normal wild type, driver: mutant) were cloned into pMD18-T vector. Two hundred SSH cDNA clones were screened by dot blot array, and 151 clones were identified as differentially expressed cDNA fragments in Cr3529 line. Thirty-six positive clones which showed marked expression differences were selected and sequenced. After redundant cDNAs were removed, 33 differentially expressed unique cDNA section clones were obtained. Among the 33 clones, two clones possess different parts of the cDNA sequence of the same gene coding geranylgeranyl reductase, four clones belong to unknown proteins, and the rest share homology to genes of diverse class. Sequence analysis showed that at least 12 genes were discovered to be related to the photosynthesis, seven of them coded the proteins which belong to the subunit of photosystem 2. RNA gel blot analysis showed that compared with 3529, the gene expression of the chlorophyll a /b -binding protein Lhc b2 in photosystem 2 declined markedly in the cotyledons and seedling leaves of Cr3529, indicating that the reduced light-harvesting complex 2 accumulation in thylakoid membrane of Cr3529 was due to the decrease of the related gene mRNA level for translation.Additional key words : oilseed rape, RNA gel blot analysis, sequence analysis, subtractive library.IntroductionThe Brassica napus line Cr3529 was a chlorophyll-reduced (CR) seedling mutant obtained from the doubled diploid inbred line 3529 induced by fast neutron and diethyl sulfate (Zhao et al . 2000). The chlorophyll content in the leaves of young CR seedlings was about one half of that of wild type (WT) seedlings, resulting in yellow-green cotyledons and leaves of the CR seedlings. After the five-leaf stage, the chlorophyll content of CR seedlings increased gradually as plants grew older, and the oldest leaves of mature plants had an appearance closer to that of the WT. The CR seedling trait was controlled by a pair of nuclear recessive genes and when the Cr gene was in the heterozygous condition, it had no deleterious effects on yield characteristics and disease resistance. Thus the CR trait can be used as a seedlingmarker to produce F 1 hybrids (Zhao et al . 2000, Wanget al . 2003). Compared with the wild type, the plastids in Cr3529 had fewer and smaller grana and the average number of lamellae per grana was 5.45, about one half of that of the WT (Zhao et al . 2003). The mild electrophoresis of the pigment-protein complexes of thylakoid membrane revealed that the pigment-protein composition of Cr3529 changed and the protein gel blot analysis showed that the polypeptide of the major light-harvesting complex 2 (LHC 2) of photosystem 2 (PS 2) in Cr 3529 thylakoid membrane decreased markedly (Zhao et al . 2001).In this study, we used suppression subtractive hybridization (SSH) (Diatchenko et al . 1996) to identify differentially expressed genes in young seedlings between lines Cr3529 and its WT line 3529 in order to reveal the molecular mechanism of the mutation characters.⎯⎯⎯⎯Received 31 October 2006, accepted 25 May 2007.Abbreviations : CR - chlorophyll-reduced; LHC 2 - light-harvesting complex 2; PCR - polymerase chain reaction; PS 2 - photosystem 2; SSH - suppressive subtraction hybridization; WT - wild type.Acknowledgements : We thank Dr. Q.S. Liu, professor of Sichuan University, for reviewing the manuscript. This work was supported in part by grants from the National Natural Science Foundation of China (no. 30170500 and no. 30571174). * Author for correspondence; fax: (+86) 28 85412738; e-mail: zhaoyun@SUPPRESION SUBTRACTIVE HYBRIDIZATION487Materials and methodsPlants: The line 3529 of Brassica napus L. is a homozygous diploid inbred from gynogenetic haploid development and the line Cr3529 was a CR seedling mutant obtained from the line 3529 induced by fast neutron and diethyl sulfate (Zhao et al . 2000). The line Cr3529 and its near-isogenic line wild type 3529 were cultivated in the test field or in flowerpot in Sichuan University, Chengdu, China. The sowing was during the first ten days of September. The temperature was about 25 °C, and seed germinated and seedling grew fast during this period. Forty days after sowing, the young leaves were selected for SSH from seedlings at five-leaf stage.Total RNA extraction and mRNA isolation: Fresh tissue (4 g) was put in liquid nitrogen in a mortar and grinded quickly to a fine powder. Total RNA was extracted according to the method described by Clark (1997). The total RNA was dissolved in 0.05 cm 3 ddH 2O that had been treated with diethyl pyrocarbonate. Smartsec TM plus s pectrophotometer (Bio-Rad Laboratories, Hercules, CA, USA) was used to determinate the RNA concentration and purity. The Poly(A)-RNA was purified from the total RNA by using polyATtract mRNA isolation systems III kit (Promega , Madison, Wisconsin, USA).Suppression subtractive hybridization: SSH was performed with the PCR-select cDNA subtraction kit (Clontech , Palo Alto, CA, USA) as directed by the manufacturer’s instructions. Double-stranded cDNA was prepared from 2 µg of Poly(A)+ wild type RNA (tester population) and mutant RNA (driver population). The cDNA was then digested with Rsa I. In two separate ligations, the tester cDNA was ligated to adapters 1 and 2. In the first hybridization, an excess of driver cDNA was hybridized at 68 °C for 8 h with the tester cDNA ligated to adapter 2 in reaction 2. In the second hybridization, reactions 1 and 2 were hybridized together in the presence of fresh driver cDNA at 68 °C overnight. The substractive product was amplified by PCR using oligonucleotides that were complementary to adapters 1 (5'-CTAATACGACTCACTATAGGGCTCGAGCGGCC GCCCGGGCAGGT-3' 3'-GGCCCGTCCA-5' and 2 (5'-CTAATACGACTCACTATAGGGCAGCGTGGTCG CGGCCGAGGT-3' 3'-GCCGGCTCCA-5').Polymerase chain reaction (PCR) was performed according to the following parameters: 75 °C for 5 min and 27 cycles at 94 °C for 30 s, 66 °C for 30 s, and 72 °C for 1.5 min. Then, a nested PCR was performed as follows: 12 cycles at 94 °C for 30, 66 °C for 30 s, and 72 °C for 1.5 min. The final PCR product was identified as dominantly expressed cDNA and corresponded to the gene population over expressed in the WT seedlings. In addition, the B. napus chloroplast triazine-resistance protein (psb A) gene was amplified with subtracted and unsubtracted cDNA populations as templates, and the amplified products were compared toevaluate the efficiency of cDNA subtraction by electrophoresis.Construction of the subtracted cDNA library (T/A cloning): Products of the secondary PCR from the forward subtraction were purified using E.Z.N.A cycle-pure kit (Omega Bio-tek , Doraville, GA, USA). The purified products were then ligated into a pMD-18T vector (TaKaRa Biotechnology , Dalian, China) to trans-form Escherichia coli JM109 cells. Colonies were grown on Luria-Bertani (LB) agar plates containing 50 μg cm -3 ampicillin (Amresco , Solon, OH, USA), 1 mM isopropyl-D-thiogalactopyranoside (Gibco BRL , Grand Island, NY, USA) and 80 mg 5-bromo-4-chloro-3-indolyl bd-galacto-pyranoside (Gibco BRL ). Transformation efficiency was approximately 107 colonies per 1 μg of starting DNA. Individual recombinant white colonies were picked up and grown in LB medium containing ampicillin (50 μg) on 96-well microtiter plates.Differential screening of subtracted library : Bacterial culture of white colonies was amplified directly usingPCR in a volume of 0.025 cm 3 with nested primer 1(5’-TCGAGCGGCCGCCCGGGCAGGT-3’) and nested primer 2R (5’-AGCGTG GTCGCGGCCGAGGT-3’). One mm 3 of PCR products was then dot blotted onto nylon membranes (Hybond N +). Four identical membranes with cDNA arrays in duplicate were prepared. DIG-labelled screening probes were prepared according to DIG DNA labelling and detection kit (Roche Molecular Biochemicals , Penzberg, Germany). Four DIG-labelled differential screening probes were prepared: two subtracted cDNA probes and two unsubtracted cDNA probes. Four different DIG-labelled probes were hybridized to four identical membranes. Dot blot hybridization and washing were carried out according to the manufacturer’s instructions. Result analysis and classification of differentially screened clones were performed according to the protocol recommended.cDNA sequencing and sequence analysis: The PCR products were cloned into the p MD18-T vector (TaKaRa Biotechnology , Dalian, China) and sequenced by the chain termination reaction using an automated sequencer (SEQLAB , Gottingen, Germany). The homology searches for the nucleic acid and protein were performed using the BLAST program at the National Center of Biotechnology Information.RNA gel blot analysis: Ten µg total RNA was electrophoresed on 1 % agarose-formaldehydegels, then transferred and cross-linked to nylon membrane (Boehringer Mannheim, Germany) using the standard methods (Sambrook et al . 1989), and hybridized in a solution containing 0.5 M sodium phosphate (pH 7.0), 1 mM EDTA, 1 % BSA, and 7 % SDS for 20 h at 65 °C. After hybridization, the blot was washed twice with aH.Y. WANG et al.488solution containing 0.1× SSC and 0.1 % SDS for 5 min at room temperature, followed by two washes of the same solution at 55 °C for 20 min. Hybridization was performed with the α-32P-dCTP labeled probe of selected cDNA clone by the random priming method (Sambrook et al . 1989). The blot was exposed to Kodak X-ray film between two intensifying screens at -80 °C. The auto-radiogram was developed after 100 h of exposure.ResultsQualitative analysis of RNA: Plenty of total RNA with high quality is the base for suppression subtractive hybridization. The use of the protocol described here resulted in RNA with high quality. RNA examined by electrophoresis on 1 % agarose/TAE gels showed a 28s rRNA band which was more abundant than the 18s rRNA band, indicating that little or no RNA degradation occurred during the extraction (Fig. 1). As a chloroplast itself possesses rRNAs, more than two rRNA bands were observed (Fig. 1).Fig. 1. The total RNAs isolated from young seedling leaves of 3529 and Cr3529. Total RNA was extracted according to the method described by Clark (1997).The typical A 260/A 280 absorbance ratios of the RNA ranged from 1.8 - 2.0, indicating that little or no protein contamination has occurred (Schultz et al . 1994). The A 260/A 230 ratios were greater than 1, indicating that little or no polysaccharide or polyphenol contamination existed (Schultz et al . 1994). The A 260/A 280 absorbance ratios ofFig. 2. The mRNAs isolated from young seedling leaves of 3529and Cr3529. Poly(A)-RNA was purified from total RNA by using polyATtract mRNA isolation systems III kit (Promega ).the purrified poly(A)+ RNA of both leaves were greater than 1.9. A clear smeared band with a M r of greater than 0.5 kb was present on the 1 % agarose gel (Fig. 2), indicating that the quality of the obtained poly (A)+ RNA was sufficient for further use.Evaluation of subtraction efficiency: The key to obtaining successful SSH result was to effectively eliminate uniform cDNA appearing in both testers and drivers (Diatchenko et al . 1996). The B. napus psb A gene sequence (GenBank Accession No. M36720) was analyzed for restriction sites of Rsa I. Two primers, psbA5 and psbA3, were designed to amplify a segment (466 bp) between the two Rsa I restriction sites. PCR amplification of this segment showed that it appeared after 18 cycles when using the unsubtracted tester cDNA as a template but did not appear until after 23 cycles when using the subtracted cDNA as a template (Fig. 3). This indicated that cDNA homologous to both tester and driver has been eliminated to a certain extent. The difference of the amplification patterns between subtracted and unsub-tracted cDNAs indicated a successful subtraction (Fig. 4).Fig. 3. Estimation of the subtraction efficiency by means of amplifying the Psb A gene. The B. napus psb A gene sequence (GenBank Accession No. M36720) was analyzed for restriction sites of Rsa Ⅰ. Two primers, psbA5 and psbA3, were designed to amplify a segment (466 bp) between the two Rsa Ⅰrestriction sites with subtracted and unsubtracted cDNA populations as templates, respectively. Lanes 1 to 4: the subtracted cDNA as a template; Lanes 5 to 8:the unsubtracted tester cDNA as a template; Lane 1, 5: 18 cycles; Lane 2, 6: 23 cycles; Lane 3, 7: 28 cycles; Lane 4, 8: 33 cycles.Differential screening of SSH clones: The forward secondary PCR products were cloned into pMD-18T vectors after purification and transformed into the competent cells of E. coli JM109. A total of 1000 white clones were obtained. There were 900 fully separatedSUPPRESION SUBTRACTIVE HYBRIDIZATION489Fig. 4. The second PCR products of subtractive hybridization.SSH was performed with the PCR-Select cDNA subtraction kit (Clontech ) as directed by the manufacturer’s instructions. The substractive product was amplified by PCR using oligonucleotides that were complementary to adapters 1 and ne 1: PCR products of forward subtracted hybridization; Lane 2: PCR products of forward unsubtracted hybridization; Lane 3: PCR products of reverse subtracted hybridization; Lane 4: PCR products of reverse unsubtracted hybridization.positive clones that were selected for further PCR amplification. The results revealed that the clones had different sizes of cDNA inserts (ranged from 200 - 1000 bp) (Fig. 5).Two hundred positive clones confirmed by PCR amplification were randomly selected and screened by dot-blotting analysis. This experiment was repeated once to avoid obtaining pseudo positive result. Partial hybridization results with forward and reverse subtracted probes were shown in Fig. 6. Finally, 151 clones potentially overexpressed in wild type were screened out.Sequence data analysis: Among the 151 positive clones, 36 clones which showed the marked expression differences were selected to have their inserts sequenced and Blast searches for the 36 clones were performed. After removing redundant cDNAs, 33 differentially expressed unique cDNA section clones were obtained. Among the 33 clones, D5 and D22 possess different parts of the cDNA sequence of the same gene codingTable 1. Features of forward subtraction clones and results of BLAST search.Code CloneSize [bp ] DNA homologyAccession number Identities (aa/aa) 1 A26 173 carnitine/acylcarnitine translocase-like protein (A. thaliana ) AAM97040 35/48 (72 %) 2 A44 737 chlorophyll a/b-binding protein CP26 in PS 2 (B. juncea ) CAA65042 160/177 (90 %) 3 A47 224 putative alanine aminotransferase (A. thaliana ) AAK25905 74/75 (98 %) 4 B5 206PS 2 protein W homolog T5F17.110 (A. thaliana ) T10660 37/39 (94 %) 5 B29 236 neoxanthin cleavage enzyme-like protein (A. thaliana ) CAA16706 51/54 (94 %) 6 B44 222 unknown protein (A. thaliana ) AAL36158 46/51 (90 %) 7 B50/C22 291 signal recognition particle receptor beta subunit-like protein (A. thaliana )BAB09661 75/86 (87 %) 8 B51 170 plastidic glutamate--ammonia ligase precursor (B. napus ) CAA51280 56/56 (100 %) 9 B52 335 elongation factor 1 alpha subunit [Malusx domestica ) CAA11705 103/106 (97 %) 10 B54/C11 258 calcium-binding protein, putative (A. thaliana ) AAK76479 37/38 (97 %) 11 C2 181 unknown protein (A. thaliana ) AAL91144 56/59 (94 %) 12 C3 144 unknown protein (A. thaliana ) AAB87114 25/28 (89 %) 13 C9/C47 198 LHC 2 Type III chlorophyll a /b binding protein (B. napus ) CAA43802 44/44 (100 %) 14 C16 601 PS 2 32 kDa protein (Bryum coronatum ) AAN85803 170/187 (91 %) 15 C17 196 hypothetical protein (Guillardia theta ) AAK39727 14/30 (47 %) 16 D5 162geranylgeranyl reductase (A. thaliana ) CAA74372 52/53 (98 %) 17 D12 189 uridylyltransferase-related (A. thaliana ) NP_564010 58/62 (93 %) 18 D19 480 PS 2 reaction center W (PsbW) protein-related (A. thaliana )NP_180615 79/120 (65 %) 19 D22 172 geranylgeranyl reductase (A. thaliana ) CAA74372 56/57 (98 %) 20 D23 257 stress enhanced protein 1 (SEP1) (A. thaliana ) NP_567958 36/59 (61 %) 21 D28 333 chitinase-like protein 1 (CTL1) (A. thaliana ) NP_172076 105/110 (95 %) 22 D30 122 dehydration-induced protein (ERD15) (A. thaliana ) NP_181674 17/19 (89 %) 23 D34 134chlorophyll a/b-binding protein-like (A. thaliana ) CAB39787 16/19 (84 %) 24 D42 185 40S ribosomal protein S29 (RPS29A) (A. thaliana ) NP_189984 49/54 (90 %) 25 E7 315 serine hydroxymethyl transferase (A. thaliana ) CAB71289 96/101 (95 %) 26 E10 189 plasma membrane intrinsic protein 2 (B. napus ) AAD39374 60/62 (96 %) 27 E11 61 2C-methyl-D-erythritol 2,4-cyclodiphosphate synthase, putative (A. thaliana )NP_850971 16/19 (84 %) 28 E33 169 S-adenosyl L-homocystein hydrolase (A. thaliana ) AAL24370 31/52 (59 %) 29 E39 114fructose-1,6-bisphosphatase precursor (B. napus ) AAD12243 37/37 (100 %) 30 E40 127 succinate dehydrogenase cytochrome b subunit family protein (A. thaliana )NP_196522 35/42 (83 %) 31 E44 232 chlorophyll a/b-binding protein Lhc b2 (A. thaliana ) T52322 64/68 (94 %) 32 E46 303 vacuolar ATP synthase 16 kDa proteolipid subunit 5 (A thaliana )NP_179244 63/76 (82 %) 33E53266 40S ribosomal protein S26 homolog (A. thaliana ) CAB8743357/72 (79 %)H.Y. WANG et al.490Fig. 5. The insets of 14 randomly selected clones in forward subtracted library. Products of the secondary PCR from the forward subtraction were purified and ligated into a pMD-18T vector to transform E. coli JM109 cells.Bacterial culture of white colonies was amplified directly using PCR in a volume of 0.025 cm3 with nested primer 1 (5’-TCGAGCGGCCGCCCGGGCAGGT-3’) and nested primer 2R (5’-AGCGTG GTCGCGGCCGAGGT-3’).Fig. 6. The positive clones confirmed by PCR amplification in forward subtracted library were dot blot analyzed. DIG-labeledscreening probes were prepared according to DIG DNA labeling and detection kit (Roche Molecular Biochemicals). 1 mm3 of PCR products in forward subtracted library was dot blotted onto nylon membranes (Hybond-N+) and hybridized with forward subtracted cDNA probe and reverse subtracted cDNA probe respectively.Fig. 7. RNA gel blot verification of Lhc b2 in cotyledons and seedling leaves of Cr3529 and 3529. 10 µg of total RNAs isolated from cotyledons and seedling leaves of Cr3529 and 3529 respectively were separated in a formaldehyde gel and blotted onto a Hybond N+ membrane. RNA gel blotting was used to hybridize with the probe, the clone E44 fragment labeled with the α-32P-dCTP by the random priming method. The cotyledons were germinated for six days and seedling leaves were at five-leaves stage. geranylgeranyl reductase, four clones belonged to unknown proteins (B44, C2, C3, C17), and the rest of the clones shared homology to diverse classes of genes withB. napus, B. juncea, Arabidopsis thaliana, Malus ×domestica, Bryum coronatum and Guillardia theta in GenBank (Table 1). Sequence analysis showed that at least 12 genes were discovered to be related to the photosynthesis, seven of them coded the proteins which belonged to the subunit of PS 2, i.e. chlorophyll a/b-binding protein CP 26 (A44), PS 2 protein W homolog T5F17.110 (B5), LHC 2 Type III chlorophyll a/b binding protein (C9/47), PS 2 32 kDa protein (C16), PS 2 reaction center W protein-related (D19), chlorophyll a/b-binding protein-like (D34) and chlorophyll a/b-binding protein Lhcb2 (E44). D5 and D22 clones were homologous to a putative chl P gene (geranyl-geranyl reductase) which was supposed to be related to the synthesis of chlorophyll (Addlesee et al. 1996, Tanaka et al. 1999).SUPPRESION SUBTRACTIVE HYBRIDIZATION491RNA gel blot analysis: The gene expression of the chlorophyll a /b -binding protein Lhcb2 which corresponds to the clone E44 codes was examined in cotyledons and seedling leaves of 3529 and Cr3529 by RNA gel blot analysis. The cDNA fragment of the clone E44 labeled with α-32P-dCTP was used as a probe in hybridization of equal amounts of total RNA from cotyledons germinated for 6 d and seedling leaves at five-leaf stage, respectively. The RNA gel blot showed that the signal intensity in 3529 is much stronger than that in Cr3529 either in cotyledons or in seedling leaves and that the signal intensity in cotyledons is quite strong compared to that of leaves in both Cr3529 and 3529 (Fig. 7).DiscussionAs the result of a single gene mutation occurred in Cr3529, chloroplast structure and most growth characteristics changed (Zhao et al . 2000, 2001, 2003). In this study, SSH results showed that the expressions of many genes were reduced in Cr3529 and some down-regulated genes were related to the chloroplast development and the process of photosynthesis which might cause the change of the chloroplast structure and growth characteristics, which result in the low seed yield of the line Cr3529.It was found that the composition of the pigment-protein of Cr3529 was changed and the content of LHC 2 in Cr3529 was about one-third of that in the wild type (Zhao et al . 2001). The result of SSH showed that 7 down-regulated genes in Cr3529 belonged to the subunit of PS 2. RNA gel blot result confirmed that the gene expression of the chlorophyll a /b -binding protein Lhcb2 coding the sub unit protein of LHC 2 was reduced markedly in Cr3529. These results indicated that the reduced LHC 2 accumulation in thylakoid membrane of Cr3529 was due to a decrease level of the mRNA of the related gene for translation.The enzyme geranyl-geranyl reductase (Chl P), which the clone D5 and D22 encode, catalyzes the reduction of free geranyl-geranyl diphosphate to phytyl diphosphate, providing the side chain to chlorophylls, tocopherols and plastoquinones (Addlesee et al . 1996, Tanaka et al . 1999). As a chemical singlet oxygen quencher, α-tocopherol can protect thylakoid membranes against photodestruction through lipid peroxidation and maintain PS 2 structure and function (Trebst et al . 2002, Havaux et al . 2005). The chl P gene is up-regulated during etioplast to chloroplast and chloroplast to chromoplast development (Keller et al . 1998). In transgenic tobacco plants expressing antisense RNA for geranyl-geranyl reductase, the reduced activity of geranyl-geranyl reductase leads to loss of chlorophyll and tocopherol (Tanaka et al . 1999, Havaux et al . 2003). The phenotype of transgenic tobacco plants was similar to that of Cr3529. Therefore, the expression reduction of the chl P gene in Cr3529 may be one of the factors causing fewer and smaller grana in chloroplast and the reduced chlorophyll content in Cr 3529 leaves.Besides the photosynthesis, the down-regulated genes in Cr3529 are related to many aspects of plant cells such as signal transduction, the metabolism of amino acids, saccharides and fatty acids, secondary metabolites, stress resistance, etc . Our data suggest that, although the mutation was on a single gene locus, the CR mutation causes changes of a series of genes at the transcription level and affect various metabolism processes in Cr3529.ReferencesAddlesee, H.A., Gibson, L.C.D., Jensen, P.E., Hunter, C.N.:Cloning sequencing and functional assignment of the chlorophyll biosynthesis gene chl P of Synechocystis sp. PCC 6803. - FEBS Lett. 389: 126-130, 1996.Clark, M.S.: Plant Molecular Biology – A Laboratory Manual. -Springer-Verlag, Berlin - Heidelberg 1997.Diatchenko, L., Chris, L.Y., Campbell, A.P., Chenchi, A.,Moqadam, F., Huang, B., Lukyanov, S., Lukyanov, K., Gurskaya, N., Sverdlov, E.D., Siebert, P.D.: Suppression subtractive hybridization: a method for generation differentially regulated or tissue-specific cDNA probes and libraries. - Proc. nat. Acad. Sci. USA 93: 6025-6039, 1996. Keller, Y., Bouvier, F.D., Harlingue, A.: Metaboliccompartmentation of plastid prenyllipid biosynthesis: evidence for the involvement of a multifunctional CHL P. - Eur. J. Biochem. 251: 413-417, 1998.Havaux, M., Eymery, F., Porfirova, S., Rey, P., Dörmann, P.:Vitamin E protects against photoinhibition and photooxidative stress in Arabidopsis thaliana. - Plant Cell 17: 3451-3469, 2005.Havaux, M., Lutz, C., Grimm, B.: Chloroplast membranephotostability in chlP transgenic tobacco plants deficient in tocopherols. - Plant Physiol. 132: 300-310, 2003.Sambrook, J., Fritsch, E.F., Maniatis, T.: Molecular Cloning: aLaboratory Manual. 2nd Ed. - Cold Spring Harbor Laboratory Press, Cold Spring Harbor 1989.Schultz, D.J., Graig, R., Cox-Foster, D.L., Mumma, R.O.,Medford, J.I.: RNA isolation from recalcitrant plant tissue. - Plant mol. Biol. Rep. 12: 310-316, 1994.Tanaka, R., Oster, U., Kruse, E., Rudiger, W., Grimm, B.:Reduced activity of geranylgeranyl reductase leads to loss of chlorophyll and tocopherol and to partially geranylgeranylated chlorophyll in transgenic tobacco plants expressing antisense RNA for geranylgeranyl reductase. - Plant Physiol. 120: 695-704, 1999.Trebst, A., Depka, B., Holländer-Czytko, H.: A specific role fortocopherol and of chemical singlet oxygen quenchers in the maintenance of photosystem 2 structure and function in Chlamydomonas reinhardtii . - FEBS Lett. 56: 156-160, 2002.Wang, M.L., Zhao, Y.: Breeding a genic and cytoplasmicdouble-MS line with Cr marker in Brassica napus . - J.H.Y. WANG et al.492Sichuan Univ. 40: 978-981, 2003.Zhao, Y., Du, L.F., Yang, S.H., Li, S.C., Zhang, Y.Z.:Chloroplast composition and structural differences in a chlorophyll-reduced mutant of oilseed rape seedlings. - Acta bot. sin. 43: 877-880, 2001.Zhao, Y., Wang, M.L., Li, J., Zhang, Y.Z.: Observation of thechloroplast in chlorophyll-reduced seedling mutant Cr 3529, Brassica napus L. - J. Sichuan Univ. 40: 974-977, 2003. Zhao, Y., Wang, M.L., Zhang, Y.Z., Du, L.F., Pan, T.: Achlorophyll-reduced seedling mutant in oilseed rape, Brassica napus , for utilization in F 1 hybrid production. - Plant Breed. 119: 131-135, 2000.Kays, S.J., Nottingham, S.F.: Biology and Chemistry of Jerusalem Artichoke Helianthus tuberosus L. - CRC Press, Taylor and Francis Group, Boca Raton - Abingdon - Oxon - New York 2008. 478 pp. ISBN 10-1-4200-4495-8Jerusalem artichoke (Helianthus tuberosus L.) is one of rich plant sources for inulin and other fructooligo-saccharides {as e.g . yacon [Smallanthus sonchifolius (Poepp. et Endl.) H. Robinson ]} that may provide dietary health benefits for obesity, diabetes, and several other health issues and with its possible use for biofuels is drawing tremendous recent interest. With its ready cultivation and minimal pest and disease problems, Jerusalem artichoke is an underutilized resource that possesses the potential to meet major health and energy challenges. “Biology and Chemistry of Jerusalem Artichoke” is a comprehensive, up-to-date book, which presents the unique biological and chemical properties of this crop. Citing a diverse cross-section of references, it reviews the history, classification, morphology, and anatomy of the plant. It details inulin chemistry addressing its properties and structure, extraction, and modification using microbes, enzymes, and a wide range of chemical processes. The book examines the use of Jerusalem artichokes as a biofuel and the role of inulin derived from the crop in combating obesity and diabetes, as well as promoting bone, blood, bowel, and immune health. A comprehensive chapter addresses genetic resources, breeding, breeding methods, hybridization, and the heritability of important traits. The book details developmental biology in terms of maximizing yield and determining resource allocation as well as controlling pests and disease. It concludes with practical information on agronomic methods, storage, the economics of crop production, and future prospects for utilization. This book provides the most comprehensive resource to date on this extremely useful crop and could serve as a valuable single reference source. The book focuses on Jerusalem artichoke as a source of inulin production and presents an up-to-date review of research on inulin and other fructooligosaccharides (FOS), and their derivatives with healthy and beneficial effects against diabetes and obesity. The authors provide industrial applications of Jerusalem artichoke, as well as its use as a feedstock for the production of biofuel. They address genetic resources, breeding, and the heritability of important traits and explain developmental biology within the context of maximal yield and resource allocation as well as offer information on agronomic methods, storage, economics, and future prospects for utilization. The book is divided into fourteen chapters detail dealing with all aspects of this crop – beginning from its nomenclature, identification, origin and history, distribution, following morphological differences between cultivars and clones. Significant chapter is devoted to tuber chemical composition, inulin and FOS chemistry, methods of their isolation, extraction, purification, fractionation, analysis, drying and storage, sources of insulin, uses for native and fractionated insulin, microbial and enzymatic modifi-cation of insulin, chemical modification of inulin and value in human and animal diet. In the other part the use of biofuel biomass, direct combustion, and biological conversion is discussed. Other chapters inform a reader about genetic resources, breeding and cultivars, breeding programs, cytology, interspecific hybrids, controlled crosses, traditional breeding, breeding techniques, flowering time, manipulation, irradiation, selection criteria, selection sequence, transgenic plants, genetic resources, molecular genetics, cultivars and clones. There is also in detail described fructan metabolism, additional metabolic pathways, yield, growth analysis and modelling, environmental factors affecting yield, agronomic practices, planting date, planting, weed control, fertili-zation, irrigation, harvesting and handling, as well as different pests and diseases, insect pests, molluscs, nematodes and other pests, fungal, bacterial and viral diseases characteristic for the Jerusalem artichoke. On the basis of 25 years running of their own research work with this plant and an extensive bibliography, the authors discuss propagation, tubers, rhizomes, tissue culture, slips, cuttings, seed developmental biology, resource allocation, and yield, developmental stages, photo-synthesis, respiration, assimilate allocation strategy, carbon transport, sink strength in relation to allocation, assimilate allocation and redistribution, storage options, storage losses and alterations in composition during storage, controlled atmosphere storage, effect of irradiation and economics and future prospects for artichoke utilizing. Thus, this book could be a very useful source of up-to-date information for both, experimental botanists, biochemists and physiologists, as well as for specialists, who are interested in the breeding, cultivation and many-sided utilization of this crop.J. LACHMAN (Prague )。
【国家自然科学基金】_抑制差减杂交(ssh)_基金支持热词逐年推荐_【万方软件创新助手】_20140801
推荐指数 2 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1
2010年 序号 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32
科研热词 推荐指数 抑制差减杂交 5 表达序列标签 3 低温 2 马铃薯 1 酵母双杂交 1 辣椒 1 茎线虫 1 茄子 1 芝麻 1 肺发育 1 耐湿性 1 紫外线胁迫 1 类赖氨酰氧化酶-1(loxl-1) 1 甘薯 1 桃蚜 1 新疆野苹果 1 抑制性差减杂交(ssh) 1 抑制性差减杂交 1 干旱胁迫 1 差异表达基因 1 差异表达 1 差减杂交库 1 实时荧光定量pcr
推荐指数 4 2 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1
2009年 序号 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17
科研热词 差异表达基因 褐飞虱 表达 糖代谢 瓯江彩鲤 水稻品种抗性 水稻 氨基转移酶 条锈菌 斑点杂交 抑制性差减杂交 抑制差减杂交(ssh) 小麦 基因表达 向重性 克隆 体色
2011年 序号 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27
2011年 科研热词 推荐指数 抑制差减杂交 5 est 2 雄性不育 1 野生大豆 1 茶树 1 茶尺蠖 1 茄子 1 致病相关基因 1 罗汉果 1 约氏疟原虫 1 甜苷v 1 温敏单性结实 1 混合虫体 1 氮缺乏 1 松材线虫 1 昆虫取食 1 按蚊 1 抑制消减杂交 1 抑制差减杂交技术(ssh) 1 微茫藻 1 大豆花叶病 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1
利用抑制性差减杂交技术筛选草原龙胆花器官发育特性基因
［ 文献 标 识 码 】 Ａ
Ｐ ｒ ｉ ｍａ ｒ ｙ Ｓ ｃ ｒ ｅ ｅ ｎ ｉ ｎ ｇ ｏ ｆ Ｈｏ ｍｅ ｏ ｔ ｉ ｃ Ｇｅ ｎ ｅ ｓ Ｄｕ ｒ ｉ ｎ ｇ Ｆ ｌ ｏ ｒ ａ ｌ Ｄｅ ｖ ｅ ｌ ｏ ｐ ｍｅ ｎ ｔ ｗｉ ｔ ｈ Ｓ ｕ ｐ ｐ ｒ ｅ ｓ ｓ ｉ ｏ ｎ
［ Ａｂ ｓ ｔ ｒ ａ ｃ ｔ 】 Ｏｂ ｊ ｅ ｃ ｔ ｉ ｖ ｅ ：Ｔ ｏ ａ ｎ ａ ｌ ｙ ｚ ｅ ｔ ｈ ｅ ｍ ｏ ｌ ｅ ｃ ｕ ｌ ａ ｒ ｍ ｅ ｃ ｈ ａ ｎ ｉ ｓ ｍ ｓ ｏ ｆ ｌ ｆ ｏ ｒ ａ ｌ ｄ ｅ ｖ ｅ ｌ ｏ ｐ ｍ ｅ ｎ ｔ ｉ ｎ ｌ ｉ ｓ ｉ ａ ｎ ｔ ｈ ｕ ｓ（ Ｅ ｕ ｓ ｔ ｏ ｍ ａ ｇ ｒ ａ ｎ ｄ ｉ ｌ ｆ ｏ ｒ ｕ ｍ ） ，
ｔ ｒ ａ ｃ ｔ ｉ ｖ ｅ ｈ ｙ ｂ ｉ ｒ ｄ ｉ ｚ ａ ｔ ｉ ｏ ｎ ｔ ｅ ｃ ｈ ｎ ｏ ｌ ｏ ｇ ｙ ｗ ａ ｓ ａ ｐ ｐ ｌ ｉ ｅ ｄ ｉ ｎ ｔ ｈ ｅ ｓ ｅ ａ ｒ ｃ ｈ ｏ ｆ ｆ ｌ ｏ ｒ ａ ｌ ｏ ｒ ｇ ａ ｎ ｉ ｄ ｅ ｎ ｔ ｉ ｔ ｙ ｇ ｅ ｎ ｅ ｓ ｄ ｕ ｉ ｒ ｎ ｇ ｆ ｌ ｏ ｒ ａ ｌ ｄ ｅ ｖ ｅ ｌ ｏ ｐ ｍｅ ｎ ｔ ｉ ｎ ｌ ｉ ｓ ｉ ａ ｎ ｔ ｈ ｕ ｓ ．Ａ ｓ ｕ ｂ ｔ ｒ ａ ｃ ｔ ｉ ｖ ｅ ｃ ＤＮＡ ｌ ｉ ｂ ｒ ａ ｙ ，ｗ ｒ ｈ ｉ ｃ ｈ ｗａ ｓ ｅ ｎ ｉ ｒ ｃ ｈ ｅ ｄ ｆ ｏ ｒ ｇ ｅ ｎ ｅ ｓ ｒ ｅ ｌ ａ ｔ ｅ ｄ ｔ ｏ ｌ ｆ ｏ ｗｅ ｒ ｏ ｒ ｇ ａ ｎ ｃ ｈ ａ ｒ ａ ｃ ｔ ｅ ｉ ｒ ｓ ｔ ｉ ｃ ｓ ，ｗ ａ ｓ ｃ ｏ ｎ ｓ ｔ ｒ ｕ ｃ ｔ —
１ １
差减杂交的基本原理
抑制差减杂交(suppression subtractive hybridization，SSH)技术是1996 年Diatchenko 等人发明的一种快速分离两种材料中差异表达基因的强有力技术，是以抑制PCR(suppression PCR)和差减杂交技术为基础，将标准化检测子cDNA 单链步骤和差减步骤合为一体的技术。
在众多差别表达基因的筛选方法中，以较为简单，假阳性低等众多的优点脱颖而出，近年来该技术在植物，动物差异表达基因的筛选中得到了最为广泛的应用，受到越来越多的重视。
运用差减杂交的方法可以用来进行两种材料中差异表达基因的分析，用来克隆缺失或突变的基因，差减杂交尤其较适合运用在一些基因组信息较为不清楚的物种上，从而分离到一些未知功能的新基因。
因此差减杂交可以为我们进一步研究差异基因或寻找新的功能基因提供基础。
它的主要原理是首先将两个真核生物mRNA样本均转化为cDNA，我们将需要检测的物种cDNA 设为“tester”，将作为对照的物种cDNA 设定为称为“driver”，通过两轮杂交，将表达没有差异的基因消减下去，差异的基因被大量富集，然后经过两轮抑致性PCR将差异的基因扩增出来。
首先将待检测的样品(Tester)和对照样品(Driver)中的总RNA 提取出来，分离出mRNA，然后将分离出的Tester和Driver中的总的mRNA合成为cDNA。
再把由mRNA 逆转录来的检测子cDNA(Tester)和驱动子cDNA(Driver)分别用同一种识别四碱基序列的限制性内切酶RsaI 消化。
一般选用Rsa I 或HaeIII 两种限制性内切酶对cDNA进行酶切，产生大小适当的末端为平头的片段。
识别四碱基酶切位点的RsaI内切酶是最为常用的内切酶，因为RsaI 酶切识别位点在基因组中相对较少，酶切后产生的片段较大。
因而既减少了基因组cDNA的复杂性，又提高了每个基因的代表性，这样可以形成适当长度平末端的cDNA片段。
抑制差减杂交法_SSH_及其在植物中的应用
e)才得以指数扩增 ,而 c类因一端有接头 ,另一 端无接头 ,只能线性扩增 ,形成牢固的“锅 - 柄 ” 结构 ,不能有效扩增 。第二轮 PCR 实际上为巢 式 PCR ,极大地提高了扩增的特异性 ,使得差异 表达目的基因片段大量富集 。
3 SSH 的技术评价
3. 1 主要优点 (1)假阳性率较低 。 SSH方法采用加接头和
然后各自与过量的 D river cDNA 变性后退火杂 交 ,这时得到 4种产物 a, b, c和 d。这种不充分 杂交使单链 cDNA 分子在浓度上基本相同 ,同时 由于 Tester cDNA 与 D river cDNA 的序列相同片 段大都形成异源双链分子 c,使得差异表达基因 得到第一次富集 。然后混合两份杂交样品 ,同时 加入过量的新的变性 D river cDNA 进行第二次差 减杂交 ,这次杂交进一步富集了差异表达 cDNA , 并形成了两个 5’端分别接有不同接头的双链分 子 e。杂交完全后 ,补平末端 ,加入合适引物 (即 部分接头 1和接头 2的单链寡核苷酸片段 )进行 两轮 PCR扩增 。第一次 PCR 是基于抑制反应 , 只有两端连有不同接头的双链 cDNA 片段 (如
39
山西农业科学 2007年 35卷第 4期
玉米 、小麦 、马铃薯 、大豆 、辣椒 、胡萝卜 、大麦 、棉 花 、康乃馨 、拟南芥 、甘蔗 、甜菜 、人参 、芒果 、灌 木 、梭梭 、橡胶树等多种植物 。
目前 , SSH方法在植物上的应用主要包括两 个方面 :一方面涉及植物的生长发育及组织特异 性研 究 。如 刘 军 等 [ 6 ] 以 水 稻 茎 尖 分 生 组 织 ( SAM )为对照材料 ,幼穗分生组织 ( pb / sb)为试 验材料 ,进行抑制性差减杂交 ,分离得到了 40个 水稻幼穗分生组织中特异表达或表达增强候选 基因 ; Kim M 等 [ 7 ]通过此技术分离得到了辣椒素 的生物合成相关基因 ; Feng DR 等 [ 8 ]也对水稻茎 尖分生组织和开花分裂组织的差异表达进行了 筛选 ; Kloos DU 等 [ 9 ]对甜菜根中表达的基因进行 了分离和鉴定 ; 常青山等 [ 10 ]同样应用此技术对 矮败小麦花药中特异表达的基因进行了研究 ;罗 志勇等也进行了人参皂苷生物合成相关基因的 筛选和鉴定 [11 ] ; 张雷等应用此项技术分离了胡 萝卜体细胞胚根发育相关基因 [ 12 ]等 。
软腐病菌诱导的大白菜抑制差减杂交文库构建及分析
１１７条 长 度 大 于 １０ｂ 、 质 量较 好 的 Ｅ Ｔ 序 列 。利 用 Ｄ Ａ ｔｒ． 上 述 Ｅ Ｔ 进 行 聚 类 ，共 获取 ７ ３个非 冗余 ０ ０ ｐ Ｓｓ Ｎ ｓ ５０对 ａ Ｓｓ ５
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关键 词 ：软 腐 病 ；抑 制 差 减 杂 交 ；表 达 序 列 标 签 ；大 白 菜 中图 分 类 号 ：￥ ３ ．；Ｑ ８ ６ ４１ ７ 文献标志码：Ａ 文 章 编 号 ： １０ — ３９ ２ １） ７ ０ ４ － ６ ０ ５ ９ ６ （０ Ｏ ０ － ０ ５ ０
Ｃｏ ｓ ｒ ｃ ｉｎ ａ ｄ ａ ａｙ ｉ ｏ ｎ ｔｕ ｔ ｎ ｎ ｌｓｓ ｆＳＳＨ ｌ ｒ ｒ ｆＣｈ ｎ ｓ ａ ｂ ｇ ｎ ｅ ｔ ｄ ｏ ｉ ａ ｙ ｏ ｉｅ ｅ ｃ ｂ ａ ｅ ｉｆ ｃ ｅ ｂ ｂ ｗｉ ｉ ａ ｏ ｏ ｏ ａ ｓ ｂ ｐ ａ ｏ ｏ ｏ ａ Ｑ ｈ ｐ ｇ ， Ａ Ｒ ｎ ｃｉ Ｃ Ｉ ｈｎ ｙ Ｅｒ ｎ ａ ｃ ｒ ｔ ｖ ｒ ｕ ｓ ．ｃ ｒ ｔ ｖ ｒ ／ ｕ Ｓ ｕｉ ’ Ｍ ｏｇ ａ ， Ｕ Ｃ ｏ ｇ ｎ一
抑制性消减杂交技术介绍
A B C
D
•
实验流程
由RNA合成 cDNA A B C D 第一次消减杂交
Rsa I 酶切
cDNA 末端接头连接
E E
第二次消减杂交 加入共用PCR引物 末端补齐 A, D: 不能被扩增 第一次PCR扩增 第二次PCR扩增 富集差异表达基因
•
C: 线性扩增 B: 扩增受到抑制
EE
5’ 3’
3’ 5’
•
Tester杂交液 （Adaptor 2R） A B C D
Driver cDNA （加热变性）
Rsa I 酶切
cDNA 末端接头连接
E
实验流程
由RNA合成 cDNA
Rsa I 酶切
A, B, C, D cDNA 末端接头连接
E
末端补齐 第一次消减杂交 第二次消减杂交 末端补齐 第一次PCR扩增 第二次PCR扩增 E E 富集差异表达基因
Driver cDNA （过量）
Tester cDNA with Adaptor 2R
A B C D
A
B
C D
•
实验流程
由RNA合成 cDNA Tester杂交液 （Adaptor 1） A B C 第一次消减杂交 D 第二次消减杂交 末端补齐 第一次PCR扩增 第二次PCR扩增 富集差异表达基因 A, B, C, D
抑制性消减杂交
主要内容
1 2 3 抑制性差减杂交技术概述
抑制性差减杂交原理 抑制性差减杂交流程
•
抑制差减杂交技术SSH
抑制差减杂交（SSH）SSH是一种 基于抑制PCR和差减杂交技术建 立的,在转录水平上研究基因表达 的技术。
•
抑制性消减杂交—原理
萘胁迫下水稻幼苗抑制差减杂交cDNA文库构建
ｔ ｎＫｔ具体操作按照说明书进行 。并参 照 Ｄａ ｈｎｏ ｉ ｉ ｏ ， ｉ ｃｅｋ ｔ
Ｌ等 方法简述 如下 ： 先把 ｍ Ｎ 首 Ｄ Ａ分别为 Ｔ ｓ ｒ Ｄ ｖｒ然后用 Ｒ ａ ｅｔ 和 ｒ ｅ， ｅ ｉ ｓ Ｉ
因表达差异提供最有效的方 法。
水稻幼苗根系作为 Ｄ ｖｒ ｉ ｒ ｅ。每一 时间处理 的材料混合 后取与对照组等重 ５ ０ｍ ０ ｇ鲜重 根系。
１２ 总 Ｒ Ａ提 取 和 ｍ Ｎ ． Ｎ Ｒ Ａ分 离 纯 化 按 照 Ｔｉ ｌ 剂 盒 （ ｉｃＢ Ｌ公 司 ） ｒｏ 试 ｚ Ｇｂｏ Ｒ 的方 法 提 取 ，
文通过与我们 日常生活紧密相关的具有一定代表性 多 环芳烃． 萘胁 迫 下 ， 建水 稻 幼 苗根 系 抑 制差 减 杂交 构
维普资讯
第２ ５卷第 ３期 ２０ ０ ８年 ６月
生 物 学 杂 志
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Ｊ ｎ，０ ８ ｕ ２０
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ｅ， ２０ ／ ｇ的幼苗为 Ｔ ｓ ｒ通过 ｅ Ｎ ｒ 以 ０ ｍｇＫ ｅｔ ， ｅ Ｄ Ａ双链 的合成及 两 次 Ｐ Ｒ扩增 ， Ｃ 富集 到 两组 处理 中差异表 达 的大 小在 ２ ０～ ０ ｂ ０ ８ ０ ｐ的 ｅ Ｎ Ｄ Ａ序列 ， Ｇ Ｍ ｐ ． ０ ｆ ｍＬ和 ｕ ９％ ， ６ 共收集 阳性克 隆 ８０ ０ ０个 ， 形 ， ＤＡ 载 体达 及分 离萘胁迫 下抗性 基 因奠定
浓 度 与完 整 性 。 １３ 抑 制 差 减 杂 交 ． 采 用 Ｃｏｔ ｈ公 司 的 Ｐ Ｒ Ｓｌ ｔ Ｍ Ｄ Ａ Ｓｂｒ ． ｌ ｅ ｎｃ Ｃ ｅ ｃ Ｔ ｃ Ｎ ｕ ｔ ｅ ｅ ａ
几种cDNA差减文库构建方法的比较
生物技术通报ＢｉｏｔｅｃｈｎｏｌｏｇｙＩｎｆｏｒｒｎａｔｉｏｎ２０００年第６期是这一方法也有其弊端：（１）差减后获得的是ｃＤＮＡ片段而非全长ｃＤＮＡ，所以构建的文库为特异性探针文库。
（２）过程繁琐，需要四轮长、短杂交。
（３）富集产物中含有一些驱动ＤＮＡ片段。
尽管有这些缺点，这一方法目前仍被广泛使用。
２．４差减抑制杂交法（ｓｓＨ）在上述酶解与Ｐ（、Ｒ技术引入差减杂交过程的基础上，ｃＬＯＮＴＥｃＨ公司、加州大学旧金山分校和俄国科学院于１９９６年合作提出了差减抑制杂交（Ｓｕｐｐｒｅｓｓ妇ｌＳｕｂｔｒａｃｔｉｖｅＨｙｂｒｉｄｉｚａｔｉ【ｍ，ＳｓＨ）方法【４｜，并于１９９８年由ｃＬＯＮＴＥｃＨ公司将其商品化，开发成ｓｓＨ法差减文库构建试剂盒。
该方法的原理是：将驱动和试验方样品ｍＲＮＡ分别反转录成ｃＤＮＡ，分别用Ｒｓａｌ或ＨａｅⅢ酶切形成平均长度在２５６ｂＤ的片段；将试验方ＤＮＡ分成两份，分别加接头ｌ和２后与驱动ＤＮＡ进行两次差减杂交。
第一次杂交时用过量的驱动ｃＤＮＡ与两种不同接头的试验方ｃＤＮＡ分别杂交，相同部分杂交形成双链而有差别部分以单链形式保留。
第一次杂交是使试验方单链均等化（Ｎ０Ⅲ１ａｌｉｚｅｄ），使原来有丰度差别的单链正）ＮＡ的相对含量基本达到一致。
这种均等化是依据杂交的二级动力学原理实现的，即丰度高的单链国ＮＡ在退火时产生杂交的速度要快于丰度低的单链ｃＤＮＡ。
杂交的结果使试验方ｃＤＮＡ中的差异表达基因得到富集。
第二次差减杂交时，是将上述试验方的两部分杂交产物混合并加入新制备的驱动山ＮＡ，试验方中带不同接头的同源ｃＥｌＮＡ片段可杂交形成双链，一端引物的序列在两次杂交后填平末端，两侧的接头可作为以后Ｐ（、Ｒ扩增的引物。
非目标序列由于其接头带有设计的反向重复序列，所以杂交退火时形成锅柄状结构而无法扩增受到抑制作用。
虽然ＳＳＨ法与上述限制性内切酶、ＰＣＲ结合的方法原理相近，但ＳＳＨ法与之相比有如下优点：（１）假阳性率被大大降低，两步杂交和两步ＰＣＲ保证了该方法可获得较高的特异性产物。
抑制性减法杂交。
两组产物另加上 新变性的drvier cDNA再次杂交，这 样就产生了两个5’端 有两个不同接头的e 型分子，这种e型分 子正是tester较 driver特异表达 cDNA，填平粘性末 端 。
(4)两轮抑制性PCR (4)两轮抑制性PCR 两轮抑制性
以两个长接头序列 分别设计成内外两对引 利用PCR原理扩增， 原理扩增， 物，利用 原理扩增 基于PCR抑制效应的存 基于 抑制效应的存 在，只有那些代表了具 有差异表达的， 有差异表达的，且两端 同时接有不同接头的双 片段（ 型分 链cDNA片段（e型分 片段 子）才能在PCR中得到 才能在 中得到 以指数扩增， 以指数扩增，
• SSH流程： 流程： 流程
cDNA合成 Rsa I 酶切 接头连接
第一次PCR
第二次 差减杂交
第一次 差减杂交
第二次PCR
初步筛选
SSH流程
• 合成DNA 将Tester分成2等份，连接不同的接头， Driver不与接头连接 2份Tester分别与Driver混合，进行第一步 差减杂交 将2份第一步差减杂交的产物未经变性就混 合，进行第二步差减杂交 抑制性PCR扩增差异表达的序列
• 缺点
（1）SSH技术对其实材 料要求多 （2）SSH技术得到的 cDNA是限制酶消化的 cDNA，不再是全场 cDNA （3）所研究材料的差异 不能太大
SSH的应用范围
• 抑制差减杂交（SSH） 在转录水平上研究 基因表达的技术,具有稳定、高效、可靠的 特点,可对生物的生长、发育、衰老、死亡 等生命过程及生物或非生物逆境胁迫对生 物所造成的影响等进行全面、系统的分析。
(3)两次消减杂交 (3)两次消减杂交
将tester cDNA分为两组，分别于其5‘端上接上 两种不同的具有一段反向末端重复序列的寡聚核 苷酸接头,两组tester cDNA样品分别与过量的 driver cDNA进行第一次杂交，得到a、b、c、d四 型分子，使原来丰度不同的单链cDNA得到大量富 集，
抑制差减杂交法克隆小麦抗病基因相关片段研究
Ｌ Ｕ Ｚ ｉ ｉｎ （ｏ ｅｅ ｆ ｉ ｃ ｎｅ Ｃ ｎ Ｍ Ｓ ｕ ｎ ｅｓｙ ｆ ｏ ｓ ｙ ｎ ｅｈ ｏ ｇ ， ｈ ｎｓａ Ｈ ｎ ｎ １０ ４ Ｉ ｈ－ ａ ｇ ｔ ｌ Ｃ ｌ ｇ ｆ Ｓ ｉ ｃ， ｅ ｔ ｏ ｔ Ｕ ｉ ｒ ｔ ｏ ｒ ｔ ｄ ｃ ｎ ｌ ｙ Ｃ ａｇｈ ， ｕ ａ ０ ０ ） ｘ ｅａ ｌ ｏＬ ｅ ｅ ｒ ｈ ｖ ｉ Ｆ ｅ ｒａ Ｔ ｏ ４
刘志 ， 超珍１ 艳２ 祥１ 曾 ， 张增
（． １ 中南林业科技大学生命科 学与技术学 院， 湖南长沙 ４０ ０ ；． １ ０４ ２中国农业科学 院作物科学研究所 ， 北京 １０ ８ ） ００ １
摘要 以 Ｙ ４／ ８０ 的 Ｆ 代 纯合 的抗病 和 感病单株 各 ｌ Ｗ６２￣ ６ １ ０株提 取 Ｄ Ａ构 建成 近等基 因池 ， 病 池为试 验 方 ， Ｎ 以抗 以感病 池 为驱动 方用差异 筛选排 除假 阳性 ， 到 ８个阳性克 隆 ；０ｔ ｍ 杂 交表明 ， 隆 Ｔ Ｈ ｌ 得 ｓｕ ｅ ｈ 克 Ｓ —具 有抗 病池特 异性 ， 为寡拷 贝或 单拷 贝序 列 ； 测序 ，Ｓ ｌ ３８ ｐ提 交 Ｇ ｎ ａｋ 且 经 Ｔ Ｈ— 长 ８ ， ｂ ｅＢ ｎ 进行 Ｂａｔ ｌ 比对 ， ｓ 没有找 到 与之 同源 的序 列 。
ｔ ｓｅ ｎ ｕ ｃ ｐｉ ｅｐ ｏ ｒｖ ｒａ ｄＳ ｅ ｔｒａ ｄｓ ｓ ｅ ｔ ｏ ｌａ ｄ ｅ ｎ ＳＨ ｂｌ ｓ ｉ ＤＮＡ ｉ ｒｒ ｓ ｏ ｓｒｃｅ Ｔｈ ｏ ｗａｄ ａ ｄ ｒ ｖ ｒｅＳ Ｈ ｒｄ ｔ ｒ ｓ ｄａ ｏ ｅｔ ｃｅ ｎ ｌｂ ａｙＷａ ｃ ｎ ｔｕ ｔｄ． ｅｆｒ ｒ ｎ ｅ ｅ ｓ Ｓ ｐ ｏ ｕｃｓｗｅｅ ｕ ｅ ｐｒｂ ｏ ｓｒｅ ｓ ｔ ｅｌ ａ ｙｔ ｅ ｖ ｈ ａｓ ｏ ｉｉｅｃｏ ｅ ． ｏ ｉ ｖ ｌｎ ｓｗｅ ｅｓ ｒｅ ｅ ｕ ｎ ｎ ｌｎ ’Ｈ— ｓｐ ｏ ｅ ｏｂ ｐ ｃａ ｏｒｓｓａ ｔｐ ｏ ｈ ｉ ｒ ｏｒｍｏ ｅｔｅｆｌｅｐ ｓｔｖ ｌｎ ｓ ８ｐ ｓｔ ｅｃｏ ｅ ｒ ｃｅ ｎ ｄｏ ｔ ｄ ｏ ｅｃｏ ｅ Ｉ ｂｒ ｉ ａ Ｓ １ｗａ ｒ ｖ ｄｔ ｅｓ ｅ ｉｌｔ ｅ ｉｔｎ ｏ ｌ ｂｙｓｕ ｈ ｒ ｙ ｒｄｉａｉｎ Ｔｈ ｅ ｅ ｃ ｆｔｅｃｏ ｅｗａ ｕ ｒｅ ｏＧｅ Ｂａ ｋｂ ｔｎ ｉ ｌｒｓ ｑ ｅ ｃ ｓ ｏ ｄ ｏ ｔ ｅｎ ｈ ｂ ｉ ｚ ｔ ， ｅｓｑｕ ｎ ｅｏ ｌｎ ｓｑ ｅ ｄ ｔ ｎ ｎ ｕ ｏｓｍｉａ ｅ ｕ ｎ ｅＷａ ｆｕｎ ． ｏ ｈ ｉ Ｋ ｅ ｒ Ｓｕ ｐ ｅ ｓｏ ｕ ｔａ ｔ ｅｈ ｂ ｉｉａｉｎ Ｗ ｈ ａ； ｓ ａｅｒ ｓｓａ ｃ ｅＢ ｙｗｏ ｄｓ ｐ ｒ ｓｉｎｓ ｂ ｒｃｉ ｙ ｒｄｚ ｔｏ ； ｖ ｅ ｔ Ｄｉｅ ｓ ｅ ｉｔ ｎ ｅｇ ｅ
利用抑制差减杂交筛选稻瘟菌继代接种菌株差异表达基因
Ｉ ｒ ｅ ｏｕ ｄ ｒ ｔｎ ｈ ｌｃ ｌｒｍｅ ｈ ｎ ｓ ｅａｅ ｏ ａ ａ ｔ ｅ ｍｕ ａｉ ｎ， ｕ ｐ ｅ ｓｏ ｕ ｔａ ｔｄ ｈ ｂ ｄ ｚ ｔ ｎ ｎ ｏ ｄ ｒｔ ｎ ｅ ｓａ ｄ ｔ ｅｍｏｅ ｕ ａ ｃ ａ ｉｍｓｒ ｌｔｄ ｔ ｄ ｐ ｉ ｔｔ ｖ ｏ ａ ｓ ｐ ｒ ｓｉ ｎ ｓ ｂｒ ｃ ｅ ｙ ｒ ｉａｉ ｉ ｏ
Ｓ ｒｅ ｉｇ ｇｎ ｓ ｅａｅ ｄ ｐｉｅｖ ｒａｉｎｉ ｇ ａ ｏｔｅｏｙａ ｂ ｐ ｒ ｓ ｏ ｂｒｃｅ ｃｅｎｎ ｅ ｅ ｌｔｄｔ ａ ａ ｔ ａ ｉｔ Ｍａ ｎ ｐ ｒ ｒｚ ｙｓ ｐ ｅｓ ｎｓ ｔａ ｔｑ ｒ ｏ ｖ ｏ ｎ ｈ ｕ ｉ ｕ ｉ
ｈｖ ｉ ｚ ｉ ｎ ｂｒｄｉａｔＯ
浙 江 农 业 学 报 Ａ ｔ Ａ ｒ ｕｔｒｅＺ ｅａ ｇｎｉ， ０ ２ ２ （ ）７ ７ ｃ ｇｉ ｌ ａ ｈ ｉｎｅｓ ２ １ ，４ １ ：１— ５ ａ ｃ ｕ ｊ ｓ
ｈ ｐ／ ｗ ｗ ｚ ｙｂ ｃ ｔ ：／ ｗ ．ｊ ｘ ．ｎ ｔ ｎ
姜华 ， 苏志杰 ， 张震 ， ．利用抑制差减杂交筛选 稻瘟菌继代接种菌株差异 表达基 因［ ］ 浙江农 业学报 ，０２，４ １ ：１ 等 Ｊ． ２ １ ２ （ ） ７
稻上继代接种 共计获 得 １８条 Ｅ Ｔ非 ６ Ｓ 冗余序列 。拼接 后 ， ｎｇｎ Ｕ ｉｅｅ的数量 为 １０个 。利用不 同数 据库 ， １ 进行 Ｂ Ａ Ｔ Ｌ Ｓ Ｘ分析 ， 得了与这些 序列 推测 获
蛋 白相 似的功能蛋 白， 析蛋 白功能注释表明这些蛋 白主要 与合成 和信号传导相关 。Ｇ 分 Ｏ分析 表 明相关 蛋 白
Ａｂ ｔ ａ ｔ Ｒｉｅ ｂ ａ ｔ ｕ ｇ ｓｉ ｎ ｆ ｈ ｓ ｉ ｏ ｔｎ ｉｅ ｐ ｔ ｏ ｅ ｓ ｗｈ ｓ ａｈ ｇ ｎ ｃｔ ｎ ｒ ｅｃ ｎ ｂ ｎ ａ ｃ ｄ ｓｒ ｃ ： ｃ ｌｓ ｎ ｕ ｓｏ ｅ ｏ ｅｍｏ ｔ ｍｐ ｒａ ｔ ｃ ａ ｈ ｇ ｎ ， ｏ ｅｐ ｔｏ ｅ ｉ ｉ ｏ ｃ ａ ｅ ｅ ｈ ｎ ｅ ｆ ｔ ｒ ｙ ｉ
抑制差减杂交技术在细菌基因差异表达研究中的应用
维普资讯
中所 需 最 小基 因 含 量 的鉴 定 以及 细 菌产 毒 株 及 非 产
形成 的异 源双 链 分 子 ， ｄ是 ｄｉｅ Ｎ ｒ ｒ Ａ单 链 及 自身 退 ｖ Ｄ
火 的 双链 分 子 。第 １ 杂 交 后 ， ｔ ｔ 两 份 杂 交 产 次 将 ｅｅ ｓｒ
物 混合 。 同 时加 上 重 新 变 性 的 ｄｉｅ Ｎ 进 行 第 ２ ｒ ｒＤ Ａ， ｖ
抑 制 差 减 杂 交 技 术 在 细 菌 基 因 差 异 表 达 研 究 中 的 应 用
中 国预 防 医学 科 学 院 流行 病 学 研 究 所 张 丽娟 综 述 阚 飙 高 守 一 审 校
摘 要 同种 细 菌基 因组 差 异及 基 因差 异 表 达 的 鉴 定 与研 究具 有 重 要 的分 子 生物 学 意 义 。 某 一 菌株 所 具 有 的 而 另 一 菌 株 所 缺 乏 的基 因可 能 决 定 菌 株 重 要 的遗 传 特 征 。抑 制 差 减 杂 交技 术是 近 年 来 新 建 立 起 来 的 一
物 基 因组 较 复 杂 ， 此 ， Ｓ 还 无 法 直 接 对 基 因 组 因 ＳＨ
通 过 两 次 的 杂 交 及 两 次 的 Ｐ Ｒ既 消 除 了 待 比 较 菌 Ｃ 的 同 源 片段 ， 使 得 特 异 差 异 片 段 进 一 步 得 到 了 有 又 效 富 集 。 克 隆从 Ｐ Ｒ获 得 的 差 减 Ｄ Ａ 片 段 建 立 差 Ｃ Ｎ
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抑制差减杂交（SSH）
抑制差减杂交技术运用了杂交二级动力学原理,即高丰度的单链cDNA在退火时产生同源杂交的速度快于低丰度的单链cDNA,在试验组(tester)和驱动组(driver)的cDNA变性后再复性的过程中,原来在丰度上有差别的单链cDNA达到均一化。
同时,由于试验组的cDNA在进行杂交之前等分的两份加有不同的接头,因此杂交时将产生5种不同类型的分子a、b、c、d和e(图1),当采用与两个不同接头序列互补的引物进行PCR扩增时,只有目标序列得到有效扩增,非目标序列因两端存在反向重复序列,退火时易产生类似”锅柄”的结构,无法与引物配对,扩增受到抑制。
抑制差减杂交(SSH)技术的主要步骤有:
(1)cDNA的合成与酶切;
(2)试验组cDNA分成两份,并与两个不同的接头连接;
(3)试验组(tester)的cDNA与过量的驱动组(driver)cDNA杂交;
(4)选择性PCR扩增与接头相连的试验组cDNA分子;
(5)克隆PCR产物,构建差减文库;
(6)筛选文库
SSH方法一般只用于两个样品的差异比较分析,样品间的差异不宜太大或太小,而对于多个样品则无能为力,这在很大程度上限制了它的应用。
提取tester RNA的时期非常关键,选择使用该方法时,首先要根据不同的研究目的确定取材的最佳时期,取材时期不当将会给试验带来意想不到的困难,或许只得到很少几个差减克隆或根本得不到克隆,或得到一些非目的克隆。
从技术本身讲,提取的tester RNA和driver RNA及从中分离的mRNA的质量、
限制酶的酶切效率、接头的连接效率、第二次PCR产物的转化效率及差减克隆的筛选方法等关系着验的成败。
另外SSH所需的起始RNA量较大,一般为几微克,对于一些珍贵稀有的不易获得足够RNA的材料要慎重使用。
SSH方法在研究植物基因的差异表达方面已得到有效应用,该方法在应用范围和深度上可进一步拓宽和加深:
(1)利用SSH方法研究生物(如细菌和真菌等)和非生物逆境(如干旱和寒冷等)对植物的影响及生物与寄主植物的互作时,可进一步提早取材时期(如逆境诱导后的6小时或更早)和缩短取材间隔,以获得植物差异表达基因的表达动态和进行有关转录因子等方面的研究,进而获得植物与逆境互作的关键点,更好地理解其互作机制,为从分子水平上定向培育抗旱、抗寒、抗病等作物提供更可靠的理论依据。
(2)将获得的一些差异表达基因片段转化为分子标记,进行标记辅助选择,进一步为育种服务。
(3)衰老、凋亡同生长、发育一样也是植物的重要生命活动过程,利用SSH研究了解作物的衰老、凋亡机制,适当有效地延长或缩短作物的生命周期,对于提高作物的生物产量和经济产量均具有重要意义。
就马铃薯育种研究工作而言,工作重点主要集中在抗逆(如抗病、抗旱等)、品质及产量等方面,利用该方法可进一步了解其机制,将所获得的相关基因转化为分子标记等,可加速马铃薯抗逆、品质改良和产量等方面的育种研究进程。
目前我们实验正在应用该项技术进行马铃薯青枯病抗性相关基因的分离工作,并拟建立病菌诱导后特定时期的基因表达谱,以初步揭示其抗性机制,为今后顺利开展马铃薯抗青枯病育种奠定基础。
(4)基因的协同表达是植物分子生物学研究的一个重要领域,SSH方法在转录水平上作为研究生物基因表达的重要方法,可为研究者展示所研究生物某一特定时期的基因表达轮廓,此领域的研究将成为今后植物分子生物学研究的一个热点。
TAKARA的PMD18-T载体可以用哪个通用引物验证？
用这两种引物：BcaBEST Sequencing Primer M13-47和BcaBEST Sequencing Primer RV-M
Vector size (bp) 2692
Cloning Site 425
LacZ α-peptide 146-475
Ampicillin resistance gene 1632~2492
pUC origin 873-1461
primer binding sites：
BcaBEST Sequencing Primer M13-47 binding site 352-375
BcaBEST Sequencing Primer RV-M binding site 484-507
在平板培养基中加入X–gal和IPTG，由于β–半乳糖苷酶的α–互补性，可以根据是否呈现白色菌落（或噬菌斑）而方便地挑选出基因重组体。