蒙古沙冬青干旱胁迫全长cDNA文库构建及序列分析

Construction and sequence analysis of a drought-induced full-length cDNA library from Ammopiptanthus mongolicus

摘要:

为了规模化分离强抗逆植物蒙古沙冬青Ammopiptanthus mongolicus (Maxim.) Cheng f.的抗旱基因并探讨其抗旱性分子机理，用SMART (Switching mechanism at 5￠-end of RNA transcript) 技术构建了其在干旱胁迫下的全长cDNA文库。原始文库滴度为1.6×107 PFU/mL，重组率为97.7％，插入片段长度多数接近和超过1 kb。对3 000个阳性克隆进行了5￠端测序和序列分析，共获得1 450条Unigene序列。在Nt、Nr和Swissprot等数据库中进行Blast搜索，结果有919条 (占63.4％) 与已知或推测功能的基因具有同源性，其余531条 (占36.6％) 为功能未知的新基因。将Unigene进行Gene Ontology (GO) 功能分类，其中与生理过程、细胞过程、结合、催化活性和细胞组分相关的基因所占比例较高，其次是与细胞器、蛋白复合体、运输和结构分子活性相关的基因，与刺激应答、基因表达调节、生理生化调节和信号转导相关的基因也占相当比例。许多有功能注释的Unigene属于抗逆相关基因，用半定量RT-PCR方法分析其中6个基因的表达变化，证明其均参与对干旱胁迫的应答反应。为进一步开展表达谱分析和克隆、鉴定抗旱基因奠定了基础。

Abstract:

Ammopiptanthus mongolicus shows very strong resistance to severe environments. To isolate drought-resistant genes and elucidate drought-resistant molecular mechanisms of the plant, we constructed a drought-induced full-length cDNA library using SMART (Switching mechanism at 5￠-end of RNA transcript) technique. The phage titer of the unamplified library was 1.6×107 PFU/mL; the recombination percentage was 97.7%; and the sizes of most cloned cDNA fragments were around 1 kb. Three thousand positive clones were randomly selected and sequenced from their 5￠ ends, and a total of 1 450 Unigenes were identified. By Blast searches against the Nt, Nr and Swissprot databases, we found that 919 Unigenes (amount to 63.4%) showed significant similarity to the annotated genes, and the remaining 531 Unigenes (amount to 36.6%) represented novel genes without any annotation. Among the functional categories of the GO (Gene Ontology) classification, the terms related to physiological process, cellular process, binding, catalytic activity and cellular components were dominant. The next abundant terms were for organelle, protein complex, transporter activity and structural molecule activity. In addition, there were a significant proportion of the terms involved in stimulus response, gene expression regulation, regulation of physiological and biochemical processes and signal transduction. Many of the annotated Unigenes were found to be related to plant resistance to abiotic stresses, and expression analyses of 6 out of these genes by semi-quantitive RT-PCR confirmed their involvements in the response of A. mongolicus to drought stress. These results laid a foundation for the expression profile analysis and the cloning and characterization of drought-resistant genes from the plant in the future.