香蕉果实采后抑制差减文库的构建与鉴定  

Construction of Differentially Expressed cDNA Library of Banana Fruit by Using Suppression Subtractive Hybridization

作　　者：徐碧玉[1] 苏伟[1] 金志强[1] 

机构地区：[1]中国热带农业科学院热带生物技术研究所,热带作物生物技术国家重点实验室,海口571101

出　　处：《分子植物育种》2005年第4期499-502,共4页Molecular Plant Breeding

基　　金：海南省高等院校优秀中青年教师科研与教学奖励基金资助。

摘　　要：抑制差减杂交技术(suppressionsubtractivehybridization,SSH),是抑制PCR与差减杂交技术相结合而产生的。其优点是在差减杂交过程中使不同丰度cDNA的拷贝数差异趋于均衡,因此不同丰度的差异表达基因都能得到有效克隆,能有效分离低丰度的差异表达基因,避免了表达序列标签分析中高丰度基因重复测序的缺点。因此SSH技术被应用于植物差异表达基因的分离。香蕉果实采后成熟过程中发生一系列复杂的生理变化,这些变化涉及到许多基因的表达。为了研究香蕉果实采后与采前在基因表达上的差异,我们首次采用抑制缩减文库的方法,以分离香蕉果实在采后表达的基因。以采后0h的香蕉果实cDNA为驱动子,对香蕉果实采后48h果实cDNA为待测子,进行抑制缩减杂交,抑制缩减杂交产物与pGEM-TEasyVecter连接,转化E.coliDH5琢,构建了抑制缩减文库。该文库共包含312个克隆,采用PCR方法对文库进行了重组子的鉴定,共有302个重组子。该文库的构建成功为深入研究香蕉果实采后基因的表达奠定了基础,为研究香蕉果实采后成熟的分子生物学机理提供了一个新的思路。Suppression subtractive hybridization (SSH) is a method produced by the combination of suppression PCR and subtractive hybridization. SSH takes an advantage in obtaining effectively low abundant cDNA clones by making the differences in copy numbers of cDNA almost be equivalent in the procession of subtractive hybridizaiton so as to avoid sequencing high repeated cDNA produced by EST. As a result, SSH has been used in isolation of differentially expressed gene from plants. A series of physiological changes takes place in postharvested banana fruit and many genes expressions are involved in those changes. In order to study the difference in gene expression between pre- and post-harvest banana fruit and isolate the differentially expressed cDNA in postharvest banana (M.AAA Group Cavendish) fruit, a differentially expressed cDNA library of postharvested banana fruit was firstly constructed by using suppression subtractive in this study. Subtractive hybridization was perfomed by using cDNA from prehavest banana fruit as driver and cDNA from postharvested for 48h banana fruit as tester. The library was constructed by the ligation of poduct of subtractive hybridization and pGEM-T Easy Vecter and transformation of the ligations to E.coli DH5?琢. Totally 312 independent clones were found in the library and totally 302 recombinants were identified afterward by using PCR with specific primers provided in PCR-SelectTM cDNA Substraction Kit, Advantage 2 PCR Kit by CLONTECH Laboratories Inc. Those cDNA in the library can be further studied for the purpose of understanding the mechanism in postharvest banana fruit ripening.

关 键 词：果实采后 构建 差减文库 鉴定 差异表达基因 抑制差减杂交技术 分子生物学机理 cDNA 表达序列标签 香蕉果实 E.coli 抑制PCR SSH技术 PCR方法 基因重复 生理变化 成熟过程 基因表达 采后成熟 重组子 缩减 拷贝数 低丰度 

分 类 号：S668.1[农业科学—果树学]