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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1.6×107PFU/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。
In order to scale-up the drought-resistance genes of Ammopiptanthus mongolicus (Maxim.) Cheng f., A strong-resistant plant, and to explore its molecular mechanism of drought resistance, a molecular mechanism of drought resistance was constructed by using SMART (Switching mechanism at 5’-end of RNA transcript) Full-length cDNA library under drought stress. The original library titer was 1.6 × 107PFU / mL, the recombination rate was 97.7%, and the length of insert was mostly close to and over 1 kb. A total of 1 450 Unigene sequences were obtained from 3 000 positive clones by 5 ’end sequencing and sequence analysis. Blast search was carried out in databases such as Nt, Nr and Swissprot. As a result, 919 (63.4%) shared homology with known or putative genes, and the remaining 531 (36.6%) were unknown genes. Unigene was categorized into Gene Ontology (GO) functional categories with a high proportion of genes associated with physiological processes, cellular processes, binding, catalytic activity and cellular components, followed by cellular organelles, protein complexes, transport and structural molecular activity Related genes, and stimulation of response, gene expression regulation, physiological and biochemical regulation and signal transduction related genes also accounted for a considerable proportion. Many of the functionally annotated Unigene belonged to resistance-related genes, and the changes of 6 genes were analyzed by semi-quantitative RT-PCR. All of them were involved in the response to drought stress. This laid the foundation for further analysis of expression profiles and cloning and identification of drought-resistance genes.