羊草叶片cDNA文库的构建及部分表达序列标签的分析

采用SMART技术,以品质优良羊草“吉生一号”叶片为材料,构建了高质量cDNA文库。原始文库滴度达到10⁶cfu/mL,扩增文库滴度接近10¹¹cfu/mL。随机抽样检查结果表明,插入片断大小在0.5~3.0kb,主要集中在1kb左右,其中检测到插入片断大于1kb的占70%,最大达到2.5kb,其重组率达到98%。同时在扩增文库中检测到了羊草维生素E合成途径中的关键酶基因α-生育酚环化酶(TC)及γ-生育酚甲基转移酶(γ-TMT)的特异信号,挑选307个筛选出了285条EST序列,将得到的117条非重复序列与GenBank中已知序列比对,获得了如3-磷酸甘油醛脱氢酶、光系统Ⅱ蛋白D1、翻译起始因子蛋白、翻译延生因子蛋白、RNaseS-likeproteinprecursor蛋白等基因。羊草高质量的cDNA文库的构建为进一步从分子水平研究羊草及开发利用这一基因资源提供了条件。

Construction of a cDNA library of the leaf of Leymus chinensis and analysis of partial expressed sequence tags

Total RNA of Leymus chinensis (“Jishengyihao”) leaves was isolated by TRIZOL reagent and a cDNA library was constructed using SMART technology. The primary library had a high titer of 10⁶ cfu/mL, in which 98% of the clones were recombinant and the insert cDNAs were from 0.5 kb to 3.0 kb. The amplified library had a titer of 10¹¹ cfu/mL. The positive signals of TC and γ-TMT gene were detected by PCR of the amplified library. High quality sequences were shown by 307 of the cDNA clones.In the cDNA library, 117 clones were non-redundant, and BLAST led to the identification of several putative genes (e.g. glyceraldehyde-3-phosphate, RNase S-like protein precursor, translation elongation factor 1, translation initiation factor 1A, chloroplast psbA gene for D1 protein). This high quality cDNA library provides a useful tool for further study of the molecular mechanisms of the secondary metabolism of vitamin E and of gene expression in L. chinensis.