固氮施氏假单胞菌电子传递复合体rnf基因簇的功能鉴定  

Functional Characterization of Two rnf Gene Clusters Encoding Electron Transport Complex in Nitrogen-fixing Pseudomonas stutzeri A1501

作　　者：陆超 刘伟[2] 黄义 杨智敏[1,3] 尚立国[1] 张宏扬 战嵛华[1] 陆伟[1] 燕永亮[1] 林敏[1] LU Chao1, LIU Wei2 ,HUANG Yi1, YANG Zhimin1,3 ,SHANG Liguo1, ZHANG Hongyang1, ZHAN Yuhua1 , LU Wei1 , YAN Yongliang1, LIN Min1(1.Biotechnology Research Institute, Chinese Academy of Agricultural Sciences, Beijing 100081; 2.Institute of Plant Protection and Microbiology, Zhejiang Academy of Agricultural Sciences, Hangzhou 310021; 3.Plant Science and Technology College, Huazhong Agricuhural University, Wuhan 430070, Chin)

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 [3]华中农业大学植物科学技术学院,武汉430070

出　　处：《中国农业科技导报》2018年第4期44-51,共8页Journal of Agricultural Science and Technology

基　　金：国家自然科学基金项目(31470205;31470174和31770067);国家973计划项目(2015CB755700);广东省引进创新创业团队计划项目(2013S033)资助

摘　　要：能量供应是限制生物固氮效率的重要因素,电子传递复合体是生物固氮过程中能量产生的重要组成部分。基因组分析表明,在联合固氮菌施氏假单胞菌A1501基因组中存在两套编码电子传递复合体rnf,分别为基因簇rnf1和rnf2,其中rnf1位于固氮基因岛上,rnf2基因簇位于核心基因组上。为了明确两套电子传递体在生物固氮过程中的功能,分别构建了rnf1和rnf2基因簇的突变株并测定了相关表型。结果发现,在基本培养基中两个基因簇的突变都不影响菌体生长,可能相互之间存在着功能互补;固氮酶活性测定结果表明,位于固氮基因岛中的rnf1基因簇缺失造成固氮酶活下降80%以上,而rnf2基因簇的缺失对酶活无显著影响。进一步采用启动子-lac Z融合表达策略探究了基因簇rnf1对固氮酶结构基因nif H转录的影响,发现rnf1极性突变株中nif H的启动子转录活性仅为野生型的17.59%,推测rnf1缺失造成了固氮条件下能量产生匮乏,胞内碳氮比失衡,进而造成固氮系统表达受抑制。Energy supply is one of the most important factors restricting the efficiency of biological nitrogen fixation,and electron transfer complex is a significant component in energy production process of biological nitrogen fixation.Genome analysis has identified 2 sets of rnf gene clusters coding for electron transport complex in the chromosome of nitrogen-fixing Pseudomonas stutzeri A1501,naming rnf1 and rnf2. The rnf1 gene cluster locates in the nitrogen fixation island,and rnf1 in the core genome. To clarify the function of both electron transport complexes,polar mutants of rnf1 and rnf2 gene clusters were constructed individually. Growth curves determination revealed that mutation of each cluster did not affect the growth in minimal medium,but deletion of rnf1 gene cluster decreased the nitrogenase activity by 90% of the wild type. However deletion of rnf2 gene cluster had no significant effect on nitrogenase activity,suggesting that rnf1 might be involved in the electron transfer to nitrogenase in A1501. The transcription level of nitrogenase reductase encoding gene nif H was further determined by promoter-lac Z fusionexpression strategy. It was found that transcriptional activity of nif H reduced by 80% in rnf1 mutant,compared with that in the wild type. It was hypothesized that deletion of rnf1 might lead to energy lack,and an imbalance of the intracellular C/N ratio under nitrogen fixation conditions,finally affecting the expression of nif genes.

关 键 词：施氏假单胞菌A1501 电子传递链rnf 固氮酶活 固氮酶基因nifH 

分 类 号：Q78[生物学—分子生物学]