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用抑制差减杂交法分离和克隆梭梭幼苗受渗透胁迫诱导相关基因的cDNA片段

郭新红, 姜孝成, 潘晓玲, 戴玉池, 姜维明, 陈良碧

湖南师范大学生命科学学院.湖南,长沙 410081；新疆大学干旱生态环境研究所.新疆,乌鲁木齐 830046

摘要

以Hoagland溶液培养的梭梭幼苗(H)为对照群体,甘露醇处理的梭梭幼苗(M)为目标群体,进行抑制差减杂交。用经过H cDNA差减的M cDNA构建了一个含有大约400个独立克隆的差减文库;采用差减前的H cDNA和M cDNA以及正向/反向差减杂交后的cDNA为模板标记探针,对随机挑取的100个重组质粒进行差示筛选,获得了21个阳性候选克隆。从这些阳性候选克隆中随机挑取了8个进行Northern blot分析,证实其中3个候选克隆代表了在M中特异表达或表达增强的基因,序列分析和同源性比较表明它们与逆境胁迫有关;而另外5个候选克隆无Northern杂交信号,推测它们为低丰度转录本。

关键词： 抑制差减杂交; 梭梭幼苗; 渗透胁迫; cDNA文库; 特异表达

Isolation and cloning of cDNA related with the genes of Halonxylon ammodendron (Mey.) Bge. seedlings induced by osmotic stress by suppression subtractive hybridization

Guo Xinhong, Jiang Xiaocheng, Pan Xiaoling, Dai Yuchi, Jiang Weiming, Chen Liangbi

College of Life Science of Hunan Normal University.Changsha 410081,Hunan；China

Abstract

The results showed that the probes built from 3 candidate clones hybridized either only or more strongly with M total RNA, while the other 5 clones had no hybridization signals with H or M total RNA, which probably represented cDNA clones expressed by low-abundance transcripts in seedlings under osmotic stress. Sequences of the 3 positive cDNA clones were measured. By BLAST sequence similarity searching in Gene-Bank nucleotide sequence database, two cDNAs had the same sequence and shared a similarity of 98% to 100% with NaCl-specific cDNA other cDNA had some similarity with the cDNAs encoding senescence-associated genes from stored snow pea (Pisum sativum L. var. saccharatum) pods.

Key words: Suppression subtractive hybridization(SSH);H.ammodedron(Mey.) Bge;Seedlings;Osmotic stress;cDNA library

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引用本文：

郭新红, 姜孝成, 潘晓玲, 戴玉池, 姜维明, 陈良碧. 用抑制差减杂交法分离和克隆梭梭幼苗受渗透胁迫诱导相关基因的cDNA片段[J]. 生理学报 2001; 53 (5): .

Guo Xinhong, Jiang Xiaocheng, Pan Xiaoling, Dai Yuchi, Jiang Weiming, Chen Liangbi. Isolation and cloning of cDNA related with the genes of Halonxylon ammodendron (Mey.) Bge. seedlings induced by osmotic stress by suppression subtractive hybridization. Acta Physiol Sin 2001; 53 (5): (in Chinese with English abstract).