抑制性消减杂交技术介绍26页PPT
抑制性消减杂交技术筛选乙型肝炎病毒核心蛋白相互作用蛋白编码基因C1的反式调节基因
抑制性消减杂交技术筛选乙型肝炎病毒核心蛋白相互作用蛋白编码基因C1的反式调节基因【关键词】抑制性消减杂交；反式激活；克隆;，乙型肝炎病毒【Abstract】 AIM： To clone and identify human genes transactivated by human gene C1 encoding protein C1 interacting with hepatitis B virus (HBV) core antigen by constructing a cDNA subtractive library with suppression subtractive hybridization (SSH) technique. METHODS： SSH and bioinformatics techniques were used for screening and cloning of the target genes transactivated by C1 protein. The mRNA was isolated from HepG2 cells transfected with pcDNA3.1(-）C1 and pcDNA3.1(-） empty vector， respectively， SSH method was employed to analyze the differentially expressed cDNA sequence between the 2 groups. After restriction enzyme Rsa I digestion， small sizes cDNAs were obtained. Then tester cDNA was pided into 2 groups and ligated to the specific adaptor 1 and adaptor 2， respectively. After tester cDNA was hybridized with driver cDNA twice and underwent polymerase chain reaction (PCR）twice and then was subcloned into pGEMTeasy plasmid vectors to set up the subtractive library. Amplification of the library was carried out with E. coli strain DH5α. The cDNA was sequenced and analyzed in GenBank with BLAST search after PCR. RESULTS： The subtractive library of genes transactivated by C1 was constructed successfully. Colony PCR of 40 positive clones showed that these clones contained 200-1000 bp inserts. Sequence analysis was performed in 30 clones， at random， and the fulllength sequences were obtained with bioinformatics method. Altogether 18 coding sequences were gotten. CONCLUSION： The obtained sequences may be target genes transactivated by C1 among which some genes coding proteins were involved in cell cycle regulation，metabolism， tumor immunity and development， and initiation and development of liver fibrosis. This finding brought some new clues for studying the biological functions of C1.【Keywords】 suppression subtractive hybridization; transactivation; clone; hepatitis B virus【摘要】目的：筛选、克隆与乙型肝炎病毒（HBV）核心抗原(HBcAg)相互作用的蛋白基因C1的反式激活基因，探索该基因可能的生物学功能. 方法：以分子生物学技术构建C1真核表达载体pcDNA3.1(-)C1，以表达质粒pcDNA3.1(-)C1转染HepG2细胞，以空载体pcDNA3.1(-)转染的HepG2细胞为平行对照，制备转染后的细胞裂解液，提取mRNA并逆转录为cDNA，经Rsa I酶切后，将实验组cDNA分成两组，分别与两种不同的接头衔接，再与对照组cDNA进行两次消减杂交及两次抑制性聚合酶链反应（PCR），将产物与pGEMTeasy载体连接，构建cDNA消减文库，并转染大肠杆菌进行文库扩增，随机挑选阳性克隆PCR扩增后进行测序及同源性分析. 结果：成功构建人类新基因C1反式激活基因差异表达的cDNA消减文库. 文库扩增后挑选40个阳性克隆，进行菌落PCR分析，均得到200～1000 bp插入片段，随机挑选含有插入片段的30个克隆进行测序，并通过生物信息学分析获得其全长基因序列，结果共获得18种编码基因. 结论：筛选到的cDNA全长序列，包括一些与细胞周期及代谢、肿瘤发生发展及肝脂肪变及肝纤维化发生发展密切相关的蛋白编码基因，推测了C1在体内可能存在的调控机制的线索.【关键词】抑制性消减杂交；反式激活；克隆; 乙型肝炎病毒0引言乙型肝炎病毒（HBV）核心抗原（HBcAg）由乙型肝炎病毒C基因的C区编码的一种结构性蛋白，在HBV的生活周期中，HBcAg，病毒mRNA和DNA聚合酶共同构成核心颗粒，在核心颗粒中完成病毒DNA的合成. HBcAg对于乙肝病毒前基因组RNA 的装配、基因组DNA的合成具有重要作用，还与病毒成熟、识别包膜蛋白以及病毒向细胞外释放等过程密切相关［1］. HBcAg特异性CD4+ T细胞免疫应答是清除HBV的重要反应［2］. 我们利用酵母双杂交技术对白细胞cDNA文库中与HBcAg相互作用的蛋白进行研究，发现了一种能与HBcAg结合的未知功能蛋白，将其编码该蛋白的基因命名为C1，GenBank收录AY555145，利用生物信息学技术确定其开放读码框架（ORF），并对其进行了克隆化研究，顺利得到了C1基因编码序列. 其编码区为366个核苷酸(nt)，编码121个氨基酸残基，我们利用抑制性消减杂交技术（SSH）构建C1作用于肝母细胞瘤细胞系HepG2细胞的反式调节cDNA消减文库，筛选差异表达的基因片段，并应用生物信息学进行分析，为进一步了解C1的功能及其探讨HBV感染的发病机制提供理论依据.1材料和方法1.1材料HepG2细胞及感受态大肠杆菌DH5α为本室保存，pcDNA3.1（-）真核表达载体购自Invitrogen公司；FuGENE6转染试剂购自Roche公司，mRNA Purification试剂盒购自Amersham Pharmacia Biotech公司，PCRSelect cDNA Subtraction试剂盒，50×PCR Enzyme Mix， Advantage PCR Cloning试剂盒购自Clontech公司，High Pure PCR Product Purification试剂盒购自Boehringer Mannheim公司，T7，SP6通用引物及pGEMTeasy载体购自Promega公司. 真核表达质粒pcDNA3.1(-)c1由本室构建. DNA序列测定由上海联合基因公司完成.1.2方法1.2.1真核表达载体的细胞转染及mRNA提取用FuGENE6脂质体转染试剂将2 μg的 pcDNA3.1(-)c1及pcDNA3.1(-)空载体分别转染35 mm平皿HepG2细胞，48 h后收获细胞. 使用QuikPreP mico mRNA Purification试剂盒从HepG2细胞中直接提取重组表达质粒及空载体的HepG2细胞的mRNA，经琼脂糖凝胶电泳及分光光度计分别进行定量分析.1.2.2消减杂交文库的建立采用Clontech公司的PCRSelect cDNA Subtraction Kit，常规SSH方法按说明书进行：以转染了重组表达质粒及空载体的HepG2细胞mRNA为模板逆转录合成双链cDNA（dscDNA），并分别标记为Tester 和Driver，dscDNA经RsaⅠ（一种识别4碱基序列的内切酶）消化，产生相对较短的平端片段，纯化酶切产物. 将Tester的dscDNA分为两份，分别连接试剂盒提供的特殊设计的寡核苷酸接头Adapter 1和Adapter 2，然后与过量的Driver dscDNA进行杂交；合并两种杂交产物后再与Driver dscDNA作第2次杂交；然后将杂交产物做选择性PCR扩增，使Tester dscDNA中特异性表达或高表达的片段得到特异性扩增.1.2.3消减文库扩增及克隆分析扩增产物与pGEMTeasy载体连接，转化DH5α感受态细菌，在含氨苄青霉素的LB/Xgal/IPTG培养板上，37℃培养18 h. 挑取白色菌落，以pGEMTeasy载体多克隆位点两端T7/SP6引物进行菌落PCR扩增，证明含有插入片段后（200～1000 bp），随机挑选其中30个克隆增菌、测序. 应用生物信息学将测得序列GenBank数据库进行同源性分析.2结果2.1mRNA的定性、定量分析紫外分光检测显示，转染了真核表达质粒及空载体的HepG2细胞提取mRNA分别为2.50 μg和2.76 μg，A260 nm／A280 nm=2.1. 20 g/L琼脂糖凝胶电泳mRNA为大于0.5 kb清晰慧尾片状条带，证实mRNA质量完全满足进行消减杂交的要求.2.2dscDNA两端连接效率检测将连接有adaptor 1和adaptor 2的两组dscDNA 分别用不同的特异性引物（看家基因甘油三磷酸脱氢酶G3PDH引物）进行28个循环扩增，产物用20 g/L琼脂糖凝胶电泳鉴定，结果显示两组dscDNA扩增产物浓度相当，说明dscDNA已与接头高效率连接.2.3cDNA消减文库消减效率的鉴定以消减及未消减PCR产物为模板，用看家基因G3PDH引物进行PCR扩增，分别在18，23，28，33次循环结束时从体系中吸取5 μL进行电泳鉴定，结果显示，与未消减组PCR产物相比，消减组PCR产物中G3PDH基因产物大大减少，说明所构建的消减文库具有很高的消减效率(图1).2.4差异表达cDNA片段的扩增及克隆杂交产物经两轮PCR扩增后，菌落PCR扩增结果显示为200～1000 bp大小不等的插入片段，所获得的40个克隆中几乎均含有插入片段，这些条带可能代表差异表达的基因片段（图2）.M: DNA marker; 1~8: 不同克隆菌落PCR产物电泳结果.图2部分克隆菌落PCR鉴定电泳图2.5cDNA测序与同源性分析初步结果挑选30个克隆测序，与GenBank数据库进行初步比较(表1).表1阳性克隆与GenBank同源序列比较结果3讨论SSH方法是近年发展起来的一项新的基因克隆技术，与传统的方法比较，具有实验周期短、易操作、可靠性高、假阳性率低等特点，能有效地分离扩增低丰度特异表达的基因，可以在较短的时间内获得较理想的实验结果［3 ］. 我们构建真核表达载体pcDNA3.1（-）C1并转染肝母细胞瘤细胞系HepG2，以转染空白载体的相同细胞系作为对照，以2种转染的细胞系中提取的mRNA为起始材料，应用SSH方法成功地构建了C1反式激活相关基因差异表达的cDNA消减文库，随机挑选文库中30个克隆送测序进行鉴定，结果发现C1可上调一些与细胞周期及代谢、肿瘤发生发展、脂肪代谢有关的基因. 细胞周期及代谢相关蛋白，如人类假定的翻译起始因子，核糖体蛋白，均与蛋白合成有关，泛酰酸激酶（Pank）是CoA生物合成途径中的必需的酶. 肿瘤发生发展及抗肿瘤治疗相关的蛋白，如人类B细胞vrel网状内皮组织增殖病毒癌基因与多种肿瘤的发生有关，可在多种组织激活多种基因转录，如浆液性乳头状瘤、神经母细胞瘤、淋巴瘤，该基因的激活及过量表达则可能导致细胞癌变. 甲胎蛋白（AFP）参与细胞的增殖、代谢的调控以及巨噬细胞和Ｔ淋巴细胞之间的相互作用. 在免疫应答中，认为AFP的作用是免疫抑制，主要表现为抑制母体对胚胎发育的免疫应答以及肿瘤患者对肿瘤的免疫应答，因此AFP的过量表达可能抑制机体的抗肿瘤免疫，与肿瘤发生及发展密切相关［4］. 精胺合成酶与人体内精胺含量相关，精胺合成酶缺乏时，精胺及亚精胺含量减少或测不出，精胺含量与抗肿瘤药的敏感性相关. 精胺缺乏时一些抗肿瘤药［如1，3bis(2chloroethyl)Nnitrosourea］的敏感性显著增加，可见精胺缺乏有利于抗肿瘤药发挥抗肿瘤作用［5］，提示精胺合成酶的过量表达可导致对一些抗肿瘤药物的抵抗或敏感性下降. 一项在日本人群中的研究显示，PCSK9基因变异体可显著影响胆固醇和低密度胆固醇水平［6］. 枯草菌素酶在肝脏是高表达并有助于高胆固醇血症发生［7］. AFP不仅具有免疫抑制作用，运输功能也是其最基本的功能，并且与脂肪代谢有关. 已经证明其最主要的功能之一就是运输脂肪酸，大部分是不饱和脂肪酸. 资料表明，游离脂肪酸，特别是不饱和脂肪酸与酒精性和非酒精性脂肪性肝炎的发生和发展密切相关［8］. 范建高的研究提示肝组织内不饱和脂肪酸增多可通过促进产基质细胞（主要是肝细胞）增殖和（或）合成ECM，参与脂肪肝肝纤维化的形成，促进肝纤维化的发生和发展［9］. 本研究结果显示，C1可反式激活AFP及PCSK9基因，使其蛋白表达增加，提示可能与肝脂肪变及肝纤维化发生发展有关.【参考文献】［1］ Le Pogam S， Shih C. Influence of a putative intermolecular interaction between core and the preS1 domain of the large envelope protein on hepatitis B virus［J］. J Virol，2002，76:6510-6517.［2］ Riedl P， Stober D， Oehninger C， et al. Priming Th1 immunity to viral coreparticles is facilitated by trace amounts of RNA bound to its argininerich domain［J］. J Immunol， 2002， 168:4951-4959.［3］ Yang XC， Hao F， Song ZQ， et al. Identification of differentially expressed genes in anagen dermal papilla by suppression subtractive hybridization［J］. Chin Med J， 2004:117(3):371-375.［4］辛永宁，宣世英，史光军，等. 肝癌病人甲胎蛋白水平与T细胞亚群及NK活性的相关性研究［J］. 中华肝胆外科杂志，2005，11(12):851-852.［5］ Ikeguchi Y， Mackintosh CA， McCloskey DE， et al. Effect of spermine synthase on the sensitivity of cells to antitumour agents［J］. Biochem J， 2003，373:885-892.［6］ Shioji K， Mannami T， Kokubo Y， et al. Genetic variants in PCSK9 affect the cholesterol level in Japanese［J］. J Hum Genet，2004，49:109-114.［7］ Abifadel M， Varret M， Rabes JP， et al. Mutations in PCSK9 cause autosomal dominant hypercholesterolemia［J］. Nat Genet， 2003，34:154-156.［8］李夏，袁红娟，赵龙凤，等. 饱和脂肪酸及不饱和脂肪酸在大鼠脂肪性肝炎与纤维化发生中的作用［J］. 山西职工医学院学报， 2004， 14(4):1-4.［9］范建高，曾民德，洪健，等.不饱和脂肪酸对L02和HLF细胞增殖及合成细胞外基质的影响［J］. 世界华人消化杂志，1998，6:502-504.。
消减杂交
食品和化学毒物学63（2014）84-90镉诱导基因姬松茸鉴定通过抑制性消减杂交文章信息關鍵詞：食用菌,姬松茸,鎘,基因表達,抑制性消減雜交摘要:镉（Cd）是最严重的环境污染物之一。
丝状真菌是非常有前途的有机体用于控制和减少重金属的由释放的量的人力和工业活动。
然而，参与镉积累和丝状宽容的分子机制真菌不完全理解。
姬松茸，可食用蘑菇的药用价值，表明高耐受性的重金属，尤其是镉。
调查的分子机制镉暴露后姬松茸的相关反应，我们构建了正向消减文库的代表了姬松茸镉诱导基因在 4 ppm的镉胁迫对使用抑制子牵引杂交结合镜面方向选择14天。
差异筛选允许我们确定39个基因表达上调，其中26都参与了代谢，蛋白质的命运，细胞运输，运输便利化和运输路线，电池救援，国防和毒力，转录和行动用绑定功能，和13种蛋白质的编码都假定蛋白质的功能尚不清楚。
电磁炉的镉暴露后六个月姬松茸基因用RT- qPCR的进一步证实。
分离出的cDNA在这项研究有助于我们参与的生化途径，partic - ipate在丝状真菌中，以镉暴露的应答基因的认识.介绍食用担子菌姬松茸，普遍被称为“Cogumelo做索尔”在巴西，日本，'Himematsutake'，或“戎机歌”在中国，原产于巴西，和过气的小路tivated其药用用途多年在日本和中国广泛。
这种真菌被认为是一个最有价值的食用和烹饪药用蘑菇品种及其生物技术营养和药用价值也很高，有据可查（Firenzuoli等，2008）。
多糖植物复合物的姬松茸被认为是负责其免疫刺激和抗肿瘤性质（Firenzuoli等人，2008; Biedron等。
2012）。
但是，重金属的积累（尤其是镉）的姬松茸受到了关注，在过去增大而增大几几十年来由于对食品安全的负面影响，马尔萨斯潜在的威胁到消费者的健康。
的收获子实体姬松茸能积累高浓度的镉，范围从10毫克公斤1 到30毫克公斤1 干物质（卡拉克，2010年，孙等人，2012），阙比许多食用菌高得多品种包括双孢蘑菇，香菇，平菇平菇，黑木耳等。
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。
抑制性消减杂交_SSH_技术及其应用
抑制性消减杂交技术（ＳｕｐｐｒｅｓｓｉｏｎＳｕｂｔｒａｃｔｉｖｅＨｙｂｒｉｄｉｚａｔｉｏｎ，ＳＳＨ）是Ｄｉａｔｃｈｅｎｋｏ等于１９９６年依据消减杂交和抑制ＰＣＲ发展起来的一种分离差异表达基因的新方法［１，２］，该技术具有假阳性率低、筛选效率高、操作简单等优点，特别适用于克隆分析造成某种特殊表型的目的基因及其功能。
而消减文库是使用两种遗传背景相同或大致相同但在个别功能或特性上不同的材料（如同类菌种的高产、低产品系），提取ｍＲＮＡ经反转录合成ｃＤＮＡ，在一定条件下用大大过量的不含目的基因的一方作为驱动子（Ｄｒｉｖｅｒ），与含有目的基因的试验方（Ｔｅｓｔｅｒ）进行杂交，选择性的去除两部分共同基因杂交形成的复合物，最后将含有相关目的基因的未杂交部分收集后，连接到载体形成文库。
高等真核生物的所有生命现象，例如细胞生长、器官形成、恶性转化、特定代谢产物分泌等都是基因选择性表达的结果，要弄清这些生命现象的分子调节机制，就要对选择性表达的基因进行分离、克隆、序列分析，然后研究其氨基酸组成，表达产物的结构功能。
抑制性消减杂交技术为寻找表达基因和研究已知基因的新生物学功能提供了一个有利工具。
１ＳＳＨ的基本原理与过程［３，４］１．１提取样本ｍＲＮＡ，利用随机引物反转录为双链ｃＤＮＡ，将不含目的基因的一方作为驱动子（Ｄｒｉｖｅｒ），含有目的基因的样品称为试验方（Ｔｅｓｔｅｒ）。
１．２用识别四碱基的限制性内切酶ＲｓａＩ（或ＨａｅⅢ）酶切。
双链ｃＤＮＡ经酶切后，每个片段一般小于６００ｂｐ，可防止长链ｃＤＮＡ片段所形成的复杂结构干扰消减杂交。
１．３将ｔｅｓｔｅｒｃＤＮＡ分成两组（１和２），分别于其５′端接上去磷酸化的接头１和接头２（ａｄａｐｔｏｒ１，ａｄａｐｔｏｒ２）。
两接头分别是具有一段反向末端重复序列的寡核苷酸序列，由一长链（约４０余个核苷酸）和一短链（约１０余个核苷酸）组成的双链ＤＮＡ片段，长链外侧２０余个核苷酸序列与第一次ＰＣＲ引物序列相同，而内侧序列与第二次ＰＣＲ引物序列相同。
抑制性消减杂交技术的应用[1]
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・技术与方法・生物技术通报B I O TECHNOLOGY BULL ET I N2009年第5期抑制性消减杂交技术的应用李小庆 景志忠(中国农业科学院兰州兽医研究所家畜疫病病原生物学国家重点实验室农业部兽医公共卫生重点实验室甘肃省动物寄生虫病重点实验室,兰州730046) 摘 要: 基因的差异表达是调控各种生命活动的核心分子机制,而分离、克隆并进一步研究差异表达基因已成为现代分子生物学研究的热点,也是功能基因组学研究的重要内容。
在研究差异表达基因的诸多项技术中,抑制性消减杂交(SSH )技术具有特异性强、假阳性率低、灵敏度高和快速简便等优点,被广泛地应用于生命科学和医学领域的基因差异表达的研究中。
近年来,抑制性消减杂交(SSH )技术得到了相应的改进和完善,而在许多研究领域,该技术在广度和深度上都有了一些新进展。
主要就抑制性消减杂交技术的产生背景、原理、技术流程、特点及其最新应用研究进展等方面作简要综述。
关键词: 抑制性消减杂交 差异表达 功能基因 DS N 均一化技术 c DNA 微阵列Suppressi on Subtracti ve Hybr i di zati on Techn i que andProgress i n the Appli cati onL i Xiaoqing Jing Zhizhong(Lanzhou Veterinary R esearch Institute CAAS,Key Laboratory of Ani m al Parasitology of Gansu P rovince,Key Laboratory of Veterinary PublicHealth of M inistry of Agriculture,S tate Key Laboratory of Veterinary E tiological B iology,Lanzhou 730046) Ab s trac t: D ifferential exp ressi on of genes is the centralmolecular mechanis m of regulating all kinds of life acti ons 1Among all thetechnol ogies of identifying differential exp ressed genes,supp ressi on subtractive hybridizati on (SSH )is considered t o be with high s peci 2ficity,l ow backgr ound,high sensitivity,convenient mani pulati on,and had been widely app lied in life science and medicine research fields 1I n this paper,the p rinci p le,method,characteristics and the research p r ogress of SSH technique were intr oduced 1Key wo rd s: Supp ressi on subtractive hybridizati on D ifferential exp ressi on Functi onal genes DS N (dup lex 2s pecific nuclease )2nor malizati on method c DNA m icr oarray收稿日期:2008211211基金项目:国家自然科学基金项目(30871884),国家高新技术“863”项目(2006AA10A203),甘肃省支撑计划项目(0804NKCA076)作者简介:李小庆(19832),男,硕士,专业方向:畜禽疫病的分子生物学与免疫学通讯作者:景志忠,博士,研究员,主要从事病原与宿主的分子生物学与免疫学研究;E 2mail:zhizhongj@yahoo 1com 1cn 上世纪90年代以来,随着分子生物学技术的快速发展以及多物种包括人类的基因组全序列测定计划的陆续完成,分子生物学的研究热点已经从结构基因组研究转向基因功能和表达调控的功能基因组研究,于是多种用于差异表达分析的基因克隆技术相继问世。
杂交技术 PPT课件
1.印迹技术
印迹技术是指将待测核酸分子转移并结 合到一定的固相支持物上的方法。 印迹技术主要包括两个关键因素：
选择良好的固相支持物； 有效的转移方法。
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⑴ 固相支持物的选择
①具有良好的机械性能，如柔软性好，韧性 强，以便操作时不易损坏；
②具有较强的结合核酸分子的能力；结合的 稳定、牢固，能经受杂交、洗膜等操作过程， 而不会脱落或脱落极少；
特别适用于菌落原位印迹法。
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尼龙膜质地坚韧，不易损坏，操作方便， 可 进行多次重复杂交。 在低离子强度条件下，也可与核酸牢固 结合，因此适用于电转印迹法。 缺点：由于结合力强，非特异性吸附较 多，杂交信号本底较高，应注意封闭。
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⑵ 印迹方法
①斑点或狭缝印迹 ②虹吸印迹法 ③电转印迹法 ④真空转移法
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c. 将 凝 胶 浸 泡 于 适 量 的 变 性 液 中 (1.5mol/L Nacl和0.5mol/L NaOH)室温1h，其目的使 DNA变性，即将双链DNA变成单链DNA。如 果DNA片段较大(大于15kb)，可在变性前， 用稀盐酸（0.2mol/L）处理10min,因为片段 的大小直接影响转移率速。小于15Kb，转移 1h。
复性的（速度）过程的影响因素：
①DNA的浓度：DNA浓度越大、碰撞 机率越大，复性速度越快。
②DNA的分子量：大分子量的DNA扩 散速度较慢，碰撞机率较少，同时又很 难形成正确配对，因此复性速度较慢。
③温度：温度过高，有利于DNA变性 而不利于复性；而温度过低，碰撞机率 减少，易形成局部错误配对，适宜的温 度是较Tm值低15℃～25℃。
d.印迹(转移)方法：虹吸印迹法，电转印迹法 和真空印迹法。不论采用哪种方法，均是将 DNA从凝胶转移到固相支持物上
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 )。
抑制性消减杂交在生物基因克隆中的应用
抑制性消减杂交在生物基因克隆中的应用
黄凤兰;胡国富;胡宝忠
【期刊名称】《生物技术通讯》
【年(卷),期】2004(015)004
【摘要】抑制性消减杂交(SSH)是抑制PCR与消减杂交技术相结合,能对未知序列差异表达基因克隆的一种方法.该方法具有速度快、效率高、假阳性率低、目的序
列富集程度高、实验结果复杂程度低等特点.本文主要介绍其基本原理、操作过程、优缺点、在生物基因克隆中的应用,并对其应用前景进行了分析.
【总页数】3页(P396-398)
【作者】黄凤兰;胡国富;胡宝忠
【作者单位】东北农业大学,生命科学学院,哈尔滨,150030;东北农业大学,生命科学学院,哈尔滨,150030;东北农业大学,生命科学学院,哈尔滨,150030
【正文语种】中文
【中图分类】Q503;Q78
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抑制性消减杂交：生成差异调控或组织特异性cDNA探针和文库的方法
Proc. Natl. Acad. Sci. USAVol. 93, pp. 6025–6030, June 1996Biochemistry抑制性消减杂交：生成差异调控或组织特异性cDNA探针和文库的方法L UDA D IATCHENKO*, Y UN-F AI C HRIS L AU†, A ARON P. C AMPBELL†, A LEX C HENCHIK*, F AUZIAM OQADAM*, B ETTY H UANG*, S ERGEY L UKYANOV‡, K ONSTANTIN L UKYANOV‡, N ADYA G URSKAYA‡,E UGENE D. S VERDLOV‡, AND P AUL D. S IEBERT*摘要一种高效消减cDNA文库构建的新方法，抑制性消减杂交（SSH），已被开发。
它是基于最近报道的抑制性PCR，并整合了常规和消减步骤。
常规PCR步骤使所有目标cDNA的丰度都达到同一水平，消减步骤则排除了检测子与驱赶子共有的序列。
在一个标准系统中，SSH技术能够在一轮消减杂交中富集稀有cDNA达1000倍以上。
我们通过构建一个睾丸特异性cDNA文库和用消减cDNA混合物作为杂交探针鉴定人类Y染色体粘粒文库中的同源序列证实了这一技术的有效性。
人类DNA在分离粘粒的插入，进一步确证了它在睾丸中特异性表达的方式。
这一结果表明SSH技术可应用于许多分子遗传和定位克隆研究，来鉴定疾病，发育和组织特异性或差异表达的基因。
正文在高等真核细胞中，例如细胞生长，器官形成等生物学过程都受控于基因表达的不同。
要弄清调控这些过程的分子机制，必须鉴定，克隆和深入研究相关的差异表达基因（1－3）。
消减cDNA杂交是鉴定和分离差异表达基因cDNA强有力的方法。
至今，已有大量的cDNA消减方法见于报端。
概括而言，它们都涉及用一种（检测子）cDNA与另一种（驱赶子）过剩的mRNA（cDNA）杂交，然后从杂交的共有序列中分离出不杂交的片断（目标片断）的方法。
抑制消减杂交
抑制消减杂交及其在鱼类基因克隆中的应用摘要：抑制消减杂交在研究差异基因表达上表现出很大的优势，其运用在人类及高等脊椎动物中已很成熟，在对鱼类的研究中还有待进一步的发展。
关键词：鱼类；抑制消减杂交；生殖与发育基因；免疫调控相关基因1 引言随着人类基因组计划的完成及后基因组计划的启动，差异基因表达就成了一项热门的技术。
由于分子生物学及相关技术的迅猛发展，在转录水平研究差异基因表达的方法也层出不穷。
目前，研究较多的主要有mRNA差异显示技术(DDRT- PCR)、代表性差异分析技术(RDA)、基因表达系列分析技术(SAGE)、cDNA 微阵列技术(cDNA microarray)和抑制消减杂交技术(SSH)。
其中抑制消减杂交（suppression subtractive hybridization SSH）是一种将抑制PCR与消减杂交技术相结合的一种快速分离差异表达基因的方法，它将传统的消减杂交方法与抑制PCR 相结合，比较2个细胞群的mRNA 以获得差异表达的基因克隆，具有检测的特殊性，在动物、植物、微生物的生殖和发育等相关领域中得到了广泛应用。
鱼类中，抑制消减杂交的应用仅限于生殖和发育相关基因和免疫调控相关基因的研究上。
2 SSH的原理和步骤示意图2.1 SSH的原理抑制消减杂交技术是由Diatchenko等[1]于1996年以mRNA差别显示技术为基础建立起来的筛选未知差异表达基因的新技术。
它主要基于最近出现的抑制PCR ，并结合标准(normalization 或equalization ) 和消减杂交( subtractive hybridization)。
抑制PCR 通过利用含引物序列的双链接头(adaptor) 充当引物模板，使两端接上同一接头的非目的双链片段具有反向末端重复序列，PCR 中不能扩增，从而达到抑制非目的片段而选择性扩增目的片段的目的。
标准化步骤平衡了目的基因群中cDNA的丰度，即低丰度差异表达的基因不会丢失，而高丰度差异表达的基因又不会被过量分离。
抑制消减杂交技术及其在动物皮肤毛囊中的应用
抑制消减杂交技术及其在动物皮肤毛囊中的应用康晓龙;李新海;冯登侦【摘要】抑制消减杂交技术(SSH)是一种用于分离两个遗传背景相关的DNA样品之间差异表达基因的方法,在筛选组织/细胞差异表达基因方面表现出独有的技术特点.就抑制消减杂交技术的原理、步骤及动物皮肤及毛囊组织的应用进行综述.%The suppression subtractive hybridization (SSH) is widely used for separating the differential expressed gene between two genetically relative DNA sample,and shows its unique features. The paper introduces the basic principles and steps of SSH,and its applications in skin tissue and hair follicle of animal.【期刊名称】《安徽农业科学》【年(卷),期】2012(040)002【总页数】3页(P847-849)【关键词】抑制消减杂交;差异表达基因;毛囊;皮肤【作者】康晓龙;李新海;冯登侦【作者单位】宁夏大学农学院,宁夏银川750021;宁夏大学农学院,宁夏银川750021;宁夏大学农学院,宁夏银川750021【正文语种】中文【中图分类】Q7811996 年，Diatchenko等［1］和 Gurskaya等［2］建立了一种新的克隆基因的方法——抑制消减杂交法(Suppression Subtractive Hybridization，SSH），用于分离两个mRNA群体间差异表达的基因。
它克服了DDRT-PCR法的假阳性较高和RDA法消减杂交轮次较多的缺点［2－4］，非常适用于克隆分析造成某种特殊表型的目的基因及其功能研究，成为差异表达基因筛选的最有潜力的方法之一。
抑制性消减杂交技术主要包括如下几个基本步骤
抑制性消减杂交技术主要包括如下几个基本步骤：（1）双链cDNA合成与酶切：分别提取待比较的两组细胞mRNA（实验方tester、驱赶方driver），利用随机引物反转录为双链cDNA后，用四碱基识别酶如Rsa I或HindⅢ切割成平均大小为600 bp左右的平端片段；该酶约在44=256 bp处就有一个切点，一分子而来的双链cDNA经该酶酶切后，可产生数个片段，每个片段一般＜600bp，可防止长链cDNA 片段所形成的复杂结构对有效消减杂交的干扰。
（2）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、去磷酸化接头（adaptor1, adaptor2），以利于随后的选择性扩增。
接头设计特点：①接头是由一长链（约40余个核苷酸）和一短链（约10余个核苷酸）组成的一端是平端的双链DNA片段，而且双链的5′均无磷酸基团，保证了接头以唯一方向与cDNA片段连接，即接头长链的3′端与cDNA双链5′端连接；②接头长链外侧（约20余个核苷酸）序列与第一次PCR引物序列相同，内侧序列与第二次PCR引物序列相同；③在接头上含有T7启动子序列，并且含有内切酶识别位点，为以后该片段插入克隆载体提供酶切位点。
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，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。
第一次杂交中，将过量的driver cDNA分别加入两份tester cDNA中，变性后退火杂交。
第一次杂交后有4种产物a、b、c、d：a是单链tester cDNA；b是自身退火的tester cDNA 双链；c是tester和driver的异源双链；d是driver cDNA。
抑制消减杂交的原理及应用
人类染色体端粒DNA的荧光原位杂交
甘肃省肿瘤医院
抑制消减杂交的实验过程
1.探针的制备
探针是一段标记核酸序列，可与靶序列 互补形成杂交双链，通过检测杂交链信 号即可对靶序列DNA的存在及其分子大 小加以鉴别.
探针可以是克隆的，也可以是合成的。 用PCR扩增产物可制备探针。也可用提 纯的质粒进行标记制备探针
PCR-TOPO 载体(T载体的一种)
甘肃省肿瘤医院
抑制消减杂交的实验过程
※ TA克隆构建原理：
TA克隆系统由Invitrogen公司发展而来的商业性试剂盒，它用于PCR产 物的克隆和测序。其原理是利用Taq酶能够在PCR产物的3’末端加上一 个非模板依赖的A，而T载体是一种带有3’T突出端的载体，在连接酶 作用下，可以快速地、一步到位地把PCR产物直接插入到质粒载体的 多克隆位点（MCS）中。
大肠杆菌
甘肃省肿瘤医院
抑制消减杂交的实验过程
蓝白斑筛选示意图
甘肃省肿瘤医院
抑制消减杂交的实验过程
分子杂交
定义：确定单链核酸碱基序 列的技术。其基本原理是待 测单链核酸与已知序列的单 链核酸（叫做探针）间通过 碱基配对形成可检出的双螺 旋片段。这种技术可在DNA 与DNA，RNA与RNA，或 DNA与RNA之间进行，形成 DNA-DNA，RNA-RNA或 RNA-DNA等不同类型的杂 交分子。
PCR反应:杂交产物中两端连有不同接头的片段就是所寻找的差异表
达基因。经补平末端后进行巢式PCR扩增以获得所需要的差异表达基 因。
甘肃省肿瘤医院
抑制消减杂交的实验过程消减的克隆:经巢式PCR扩增得到所需要的差异表 达基因后，利用TA克隆法将 差异表达基因转化感受态大 肠杆菌，根据蓝白斑筛选原 理，筛选出转化成功的阳性 菌落。最后根据插入片段的 两端接头引物进行巢式PCR反 应对插入片段进行特异性扩 增。
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，
消减杂交技术
消减杂交技术通过对《干旱胁迫下黄檗幼苗c DNA消减文库的构建和分析》的试验的介绍，说明消减杂交技术的原理，以及技术流程。
试验摘要以干旱胁迫下的黄檗幼苗cDNA 为tester, 正常生长的黄檗幼苗cDNA为driver, 利用抑制性消减杂交技术(suppression subtractive hybridization, SSH)构建了干旱胁迫下黄檗幼苗的消减文库并对其进行了EST序列分析。
从消减文库中随机挑取20个阳性克隆, 提取质粒进行酶切和PCR鉴定, 显示文库克隆的重组率大于95%, 插入片段大小大部分集中在300~800bp之间。
随机挑取816个克隆进行测序, 得到265个基因。
将其进行同源性分析, 划分为16类。
获得了热激蛋白70、脱水响应蛋白(RD22)、通用胁迫蛋白、金属硫蛋白(MTII), 晚期胚胎丰富蛋白(LEA14)等44种与干旱胁迫相关的基因,它们涉及了植物的渗透调节、信号传递、转录调控、活性氧清除等方面。
本研究为抗逆基因克隆和系统研究干旱胁迫下黄檗基因的表达奠定了重要的理论基础。
黄檗(黄檗Phellodendron amuranse Rupr.) 又名关黄柏、黄波罗, 为芸香科黄檗属, 是第三纪古热带区系的孑遗植物。
是东北重要的珍贵阔叶树种。
从生活史看, 黄檗经历了从第三纪炎热到寒冷等一系列的气候变迁, 对自然界的非生物胁迫(如高温、干旱、寒冷)有很强的适应力。
材料与方法• 1.1 实验材料•黄檗种子采于牡丹江市, 用70%乙醇对种子表面除菌后, 4℃低温层积2个月, 播种在珍珠岩中, 25~30℃光照培养箱培养。
大约生长60 d左右对黄檗幼苗进行胁迫处理, 减小加水量使其相对水含量达到65%~70%(约6~7d), 未处理的作为对照。
处理后, 取幼苗叶片和茎干置于液氮中速冻, −70℃保存。
1.2实验方法• 1.2.1 总RNA提取和mRNA纯化•以干旱处理的黄檗幼苗为实验组, 未处理的为对照组。