抑制消减杂交法分离紫花苜蓿幼苗铝胁迫诱导表达的cDNA

Isolation and Cloning of cDNA Induced by Aluminum Stress in Alfalfa Seedling Using Suppression Subtractive Hybridization

利用抑制消减杂交(SSH)技术分离铝胁迫诱导紫花苜蓿差异表达的基因,以水培试验获取的中苜一号幼苗为材料,以80μmol/L铝离子胁迫的紫花苜蓿作为试验组,未胁迫的为驱动组,构建了一个包含456个克隆的SSH文库。对构建的文库进行鉴定,随机选取20个阳性克隆测序,共获得15条有效EST序列,然后将测序结果提交到GenBank进行Blastn比对,获得了3条未知基因的序列,推测它们可能与植物的抗铝作用有关。检测结果表明,构建的文库质量较好,可以进行进一步深入研究,为揭示植物耐铝性的分子机理提供理论基础。

The aluminum stress induced genes of alfalfa were identified by suppression subtractive hybridization（SSH） using Zhongmu NO.1 cultivar,in the experiment,half seedlings were stressed in 80 μmol/L aluminum ion stress solution for 24 h as tester,and another half seedlings were continuously cultivated in culture solution without Aluminum as driver.A subtractive cDNA library was constructed by suppression subtractive hybridization（SSH）,which was composed of approximately 456 clones.We tested cDNA library through randomly selecting and sequencing twenty clones,by which 15 ESTs were obtained.We submitted the EST sequence to NCBI,the result of which showed that three ESTs had no homology from NCBI,thus,we suggest that they might be involved in Al resistance in plant.The result showed that the constructed library performed high quality,which can be used in further study on the aluminum tolerance to provide theoretical basis of the molecular mechanism.