阿维链霉菌遗传多样性研究和aveD缺失基因的克隆.pdfVIP

摘 要 摘 要 本论文对阿维链霉菌遗传多样性和aveD缺失基因的克隆进行研究 基于目前 国内外对阿维菌素的研究现状 将基因打靶技术应用于对阿维菌素产生菌的基因 改造 并对具体的实验条件进行了初步的摸索 为敲除阿维链霉菌 aveD基因,进 一步遗传改造阿维链霉菌,构建高产工程菌 提高阿维菌素有效成分的产出奠定了 技术基础 实验的第一部分是对阿维链霉菌突变株遗传多样性的研究 选用22株经离子 束 亚硝基胍和太空搭载诱变筛选出的阿维菌素高产突变株与野生菌及出发菌株, 采用了PCR技术扩增rpsL基因,用TGGE技术对扩增结果进行分析 对它们进行了 遗传多样性分析 研究结果表明,14株菌与野生菌株4.1253对比都发生了突变 说明阿维菌素产量与rpsL基因突变有相关性 本实验的第二部分在阿维链霉菌aveD缺失基因的克隆研究中,采用开读框内 ORF定点缺失技术,根据基因库中aveD基因上下游序列,设计了PCR扩增引物, 摸索扩增条件,将纯化的aveD基因与pMD18-T连接转化E.coliDH5á,提取重组质 粒,将其命名为pHD1测序后分析aveD基因的酶切位点,发现突变株aveD基因发 生突变,丧失了Nal酶切位点中的两个切点 因此,选用BssH切除了aveD基因 中262bp的序列,获取了aveD基因内部缺失262bp的一段长为824bp的DNA片段 即aveD缺失基因,并通过转化E.coliDH5á 成功地克隆了aveD缺失基因 关键词 阿维菌素 基因打靶 温度梯度凝胶电泳 rpsL基因 -I- Abstract Abstract The research had been made on the genetic diversity and aveD deficient gene cloning of Streptomyces avermitilis. Based on the current study on avermetins, we had made a fundamental research on the idiographic experimental terms by gene targeting technology, which have great positive effects on knocking out Streptomyces avermitilis aveD gene and making further genetic modifcation for higher component productivity. The ultimate purpose of the experiment is to establish an technique for obtaining the genetical strains which produce avermectin highly. The first part of this research was about mutan t genetic diversity of Streptomyces avermitilis.22 mutants were treated by Ion bea, NTG and outer space aether induced mutation. Their genetic diversity of all these mutants were analysed through rpsL gene amplifying by PCR.TGGE results showed that 14 mutations had rpsL gene mutation. It was evident that rpsL gene mutation had some positive effect on high production strains. The secon