应用抑制差减杂交法分离玉米幼苗期叶片土壤干旱诱导的基因

Isolating Soil Drought-induced Genes from Maize Seedlings Leaves Through Suppression Subtractive Hybridization

【目的】分离土壤干旱胁迫条件下玉米幼苗期叶片诱导表达的基因。【方法】本研究利用抑制差减杂交(suppression subtractive hybridization,SSH)的方法,以耐旱自交系CN165为材料,构建了土壤干旱胁迫下玉米幼苗叶片的正向抑制差减cDNA文库。【结果】在这个文库中,随机挑取了672个阳性克隆,并进行PCR验证,对所有的单克隆进行了测序。得到了598个有效序列,经过EST聚类分析后,共得到了80个uniESTs。其中57个为contigs。23个为singlets。BLASTN的结果表明,所有的uniESTs都可以在玉米的核酸数据库中找到同源序列。BLASTX的结果表明:68个uniESTs和已知功能的蛋白有高度的相似性,8个uniESTs为未知功能蛋白和假定蛋白,4个uniESTs没有蛋白质的相似性。【结论】在这个cDNA文库中发现了大量抗旱相关的基因,这些基因涉及到植物代谢的多种途径。

[Objective] Isolation and analysis soil drought-induced genes from maize seedlings leaves. [Method] A forward cDNA library was constructed by suppression subtractive hybridization using seedling leaves of ＂CN165＂, a drought-tolerant maize inbred line. [Result]In the SSH library, 672 positive clones were picked up randomly. After PCR of each clone, all the single clones were sequenced. Totally 598 available sequences were obtained. After cluster anslysis of the ESTs sequences, 80 uniESTs were obtained, among which 57 uniESTs were contigs and 23 uniESTs were singlets. The results of BLASTN showed that all the uniESTs had homologous sequences in the nr database. The BLASTX results indicated that 68 uniESTs had significant protein homology, eight uniESTs were unknown proteins and putative proteins, and four uniESTs had no protein homology. [ Conclusion ] A large group of drought stress-induced genes were found in the cDNA library, which involved in many metabolism pathways.