施氏假单胞菌固氮调控蛋白NifA调节电子传递体rnf1基因簇的表达  

Effect of the Nitrogen Fixation Positive Regulator NifA on the Transcription of the Electron Transport Complex rnf1 Cluster in Pseudomonas stutzeri

作　　者：黄义 刘伟[2] 陆超 陆伟[1] 战嵛华[1] 张维[1] 燕永亮[1] HUANG Yi;LIU Wei;LU Chao;LU Wei;ZHAN Yuhua;ZHANG Wei;YAN Yongliang(Biotechnology Research Institute,Chinese Academy of Agricultural Sciences,Beijing 100081;Institute of Plant Protection and Microbiology,Zhejiang Academy of Agricultural Sciences,Hangzhou 310021,China)

机构地区：[1]中国农业科学院生物技术研究所,北京100081 [2]浙江省农业科学院植物保护与微生物研究所,杭州310021

出　　处：《中国农业科技导报》2018年第8期39-45,共7页Journal of Agricultural Science and Technology

基　　金：国家自然科学基金项目(31470174和31770067);国家973计划项目(2015CB755700);中央级公益性科研院所基本科研业务费专项(0392017002);广东省引进创新创业团队计划项目(2013S033)资助

摘　　要：施氏假单胞菌A1501中电子传递体基因簇rnf1位于固氮基因岛上,该基因簇的突变造成固氮酶活显著下降。在rnf1基因簇的启动子区含有固氮调控蛋白Nif A的保守结合序列,实时定量qRT-PCR分析证实了nif A突变株中rnf1基因簇的表达量与野生型相比急剧下调,暗示着Nif A直接参与rnf1基因簇的表达调控。细菌单杂交系统的体内互作实验表明,在大肠杆菌体内Nif A与rnf1基因簇启动子存在直接相互作用;进一步的凝胶阻滞试验证明原核表达纯化的Nif A蛋白与rnf1基因簇启动子序列存在体外直接结合。上述结果从分子水平上给出了两者间相互作用的直接证据,为深入研究联合固氮基因的表达调控网络奠定了基础。The electron transport complex gene cluster rnf1 was located within the nitrogen fixation island on the Pseudomonas stutzeri A1501 chromosome,and mutation of this cluster led to a sharply decrease of the nitrogenase activity. In this paper,a conserved binding site was identified in the promotor region of the rnf1 cluster. qRT-PCR results further indicated that the expression of rnf1 gene was dramatically down-regulated in the nif A mutant compared to the wild type,suggesting there was a regulatory role of Nif A to the expression of rnf1 cluster. The interaction between Nif A and the promoter region of rnf1 cluster was verified using bacteria one-hybrid system in vivo.Furthermore,the prokaryotic expression vector of nif A gene was constructed and the Nif A protein was purified,and electrophoresis mobility shift assay showed that Nif A and rnf1 promoter region could directly interact in vitro as well.Above results proved that Nif A might recognize and bind to the promoter of rnf1 cluster specifically thereby regulating its transcription,and provided basis for further studying the regulation net of nitrogen-fixing genes.

关 键 词：生物固氮 电子传递体基因簇 NIFA蛋白 凝胶阻滞试验 

分 类 号：Q78[生物学—分子生物学]