%0 Journal Article
%T 胶状溶杆菌OH17菌株遗传操作系统的构建
%A 刘琳琳
%A 钱国良
%A 刘凤权
%J 南京农业大学学报
%D 2017
%R 10.7685/jnau.201611007
%X [目的]建立胶状溶杆菌（Lysobacter gummosus）OH17菌株的遗传操作系统，通过基因重组方法来研究调控基因的功能及次级代谢产物的生物合成途径。[方法]分别将整合型突变质粒pJQ-pilR和用于同源重组的、包含参与抗菌物质生物合成的DNA片段的质粒pSEVA321-pilR，通过电转化法各自导入L.gummosus OH17之中。[结果]通过电转化获得正确转化子，结合基因重组获得突变菌株，采用PCR验证和酶切验证重组质粒的导入和目标基因pilR的缺失，进一步对突变菌株的发酵产物进行了HPLC分析，结果表明突变株合成热稳定抗真菌因子（heat-stable antifungal factor，HSAF）能力明显下降，通过将pilR回补，其合成HSAF的能力得到一定程度恢复。[结论]成功构建了L.gummosus OH17遗传操作系统，包括基因定位缺失突变系统和基因功能互补系统，为以后对OH17及其他胶状溶杆菌进行代谢产物合成及其调控基因的功能研究奠定了遗传操作基础。</br>[Objectives]This paper aims to develop the genetic system of Lysobacter gummosus OH17,and study the function of regulatory genes and the biosynthetic pathway of secondary metabolites with genetic recombination. [Methods]The integrated mutation plasmid pJQ-pilR and plasmid pSEVA321-pilR for homologous recombination which contains DNA fragment related to metabolites biosynthesis were transferred into L.gummosus OH17 by electrotransformation,respectively. [Results]The correct transformants were obtained by electrotransformation,and then the mutant strains were obtained by genetic recombination. The introduction of recombination plasmids and the deletion of target genes were verified by PCR and enzyme digestion. Further,the fermentation products of mutant strains were analysed by HPLC. The result showed that the mutants’ synthesis ability of heat-stable antifungal factor(HSAF)declined significantly,while a certain of synthetic ability could recover with the introduction of gene pilR. [Conclusions]Genetic system of L.gummosus OH17 including gene-directed mutagenesis system and gene functional complementary system were successfully developed. It laid the foundation for future genetic manipulation research such as the synthesis of metabolites and the function of regulatory genes of OH17 and other L.gummosus
%K 胶状溶杆菌
%K 遗传操作系统
%K 基因敲除
%K 基因互补&lt
%K /br&gt
%K Lysobacter gummosus
%K genetic manipulation
%K gene mutagenesis
%K gene complementary
%U http://nauxb.njau.edu.cn/oa/darticle.aspx?type=view&id=201704011