陆地棉显性无腺体近等基因系SSH文库的构建  

Construction of SSH Library from Dominant Glandless in Gossypium hirsutum L.

作　　者：朱秀兰[1] 宋国立[1] 王坤波[1] 

机构地区：[1]中国农业科学院棉花研究所农业部棉花遗传改良重点实验室,河南安阳455000

出　　处：《安徽农业科学》2009年第26期12435-12439,12502,共6页Journal of Anhui Agricultural Sciences

摘　　要：[目的]应用抑制性消减杂交技术构建差异表达cDNA消减文库。[方法]以显性无腺体中棉所12(显无中12)和中棉所12(中12)提取棉花总RNA,以显无中12为驱动子,中12为检测子,利用SSH法建立差异表达cDNA文库进行克隆,并以Nested PCR Primer 1和2R对插入片段进行PCR扩增,分别以显无中12和中12总cDNA为探针进行反向Northern杂交,筛选差异表达克隆,进行序列相似性分析。[结果]通过建立差异表达cDNA文库共获得2 280个克隆,通过反向Northern杂交共筛选363个差异表达克隆,测序后得到299个质量合格的EST序列,共56个重叠群,91个单拷贝。这些EST所涉及的基因,主要是与代谢和次生代谢调节、生物合成、细胞凋亡、信号传导等有关联的cDNAs。[结论]SSH文库的构建和分析为显性无腺体形成相关基因的克隆和结构功能研究奠定了基础。[ Objective ] The purpose was to construct differential expression cDNA subtractive library by using suppression subtractive hybrid- ization （SSH） technology. [ Method ] With CRI 12 of dominant glandless （ Xianwuzhong 12 ） and CRI 12 （ C12 ）, with Xianwuzhong 12 as driver and C12 as tester, the total cotton RNA was extracted. The clone was performed by using SSH to establish differential expression cDNA library, and the insert fragments were in PCR amplified with Nested PCR Primerl and 2R, the reverse Northern blot was done with total eDNA of Xianwuzhong 12 and C12 resp. as probes, and differential expression clones were screened to analyze the sequence similarity. [ Result] 2 280 clones were acquired by setting up differential expression cDNA library, totally 363 differential expression clones were screened through reverse Northern blot, 299 EST sequences of qualified quality, including 56 contigs and 91 single copies were obtained after sequencing. The gene involved with these EST were mainly the cDNAs that related with regulation of metabelism and secondary metabolism, biosynthesis, apop- tosis, signal transduction etc. [ Conclusion ] The construction and analysis of SSH library laid a foundation for the research of the clone and structure function of relevant gene formed by dominant glandless.

关 键 词：陆地棉 显性无腺体 抑制消减杂交 CDNA文库 

分 类 号：S562[农业科学—作物学]