Dietzia菌随机插入突变体文库的构建及功能基因筛选  

Screening functional genes from a Dietzia strain transposon-inserted mutant library

作　　者：郑鑫 王蒙 许子牧 黄晓雨 王淼啸 黄婷 方源 聂勇[2] 吴晓磊[2] 汪家权[1] ZHENG Xin;WANG Meng;XU Zimu;HUANG Xiaoyu;WANG Miaoxiao;HUANG Ting;FANG Yuan;NIE Yong;WU Xiaolei;WANG Jiaquan(School of Resources and Environmental Engineering,Hefei University of Technology,Hefei 230009,China;College of Engineering,Peking University,Beijing 100871,China)

机构地区：[1]合肥工业大学资源与环境学院,合肥230009 [2]北京大学工学院,北京100871

出　　处：《应用与环境生物学报》2020年第4期775-782,共8页Chinese Journal of Applied and Environmental Biology

基　　金：国家重点研发计划项目(2018YFA0902100,2018YFA0902103);国家自然科学基金项目(31770118,31770120)资助。

摘　　要：Dietzia属菌具有很好的烃类降解和良好的高盐、高碱耐受能力.为研究Dietzia属降解烷烃和耐受盐碱的分子机制,以Dietzia sp.DQ12-45-1b为研究菌株,利用双质粒系统p Tip-istAB-sacB和pRTSK-sacB,成功构建库容为30720的随机突变体文库.随机挑选的突变克隆及突变克隆传代20次后的Southern blot结果表明:转座序列插入的随机性较好,均为单插入突变,并且具有较好的遗传稳定性,可用于长期筛选功能基因.在此基础上,分别以高盐培养和正十六烷为单一碳源培养,利用高通量基因筛选,成功获得了一个耐盐突变体M9G和5个降解利用正十六烷相关突变株M6A、M6B、M7A、M9A、M81C.与野生型相比,5个降解利用正十六烷相关突变株的降解率下降范围为16.67%-84.35%.插入位点分析结果显示,M9G、M6A、M6B、M7A、M9A和M81C的插入基因分别预测为丝氨酸蛋白酶、铁氧化还原蛋白、假定蛋白、铁氧化还原蛋白还原酶、ABC转运蛋白和细胞色素P450类CYP153烷烃羟化酶.其中除了预测假定蛋白外,有些基因与传统上确定的功能存在差异,暗示着其可能具有新的功能.Dietzia菌随机插入突变体文库的构建及筛选可为研究Dietzia菌的抗逆和烷烃降解机制奠定基础.(图6表3参28)Dietzia bacteria can degrade a variety of n-alkanes and grow at high levels of salinity and alkalinity.Hence,it was important to construct a robust mutant library to lay the foundation for further research in the molecular mechanisms of n-alkane degradation by Dietzia bacteria as well as investigate its salt and alkali tolerance.In this study,we used the strain Dietzia sp.DQ12-45-1 b as a model as well as a dual plasmid system of p Tip-istab-sacB and prtsk-sacB to construct the transposon-inserted mutant library.We successfully constructed a transposon-inserted mutant library with a storage capacity of 30720.The southern blot analysis of the randomly selected mutants showed that they were all had single insertion mutations.Following over 20-time passages using these mutants,the inserted sequences still remained in their chromosomes,suggesting that the mutant library was robust for target gene screening.In order to explore the potential applications of this mutant library,we generated one salt tolerant mutant and five n-hexadecane degradation-related mutants of DQ12-45-1 b using high-throughput screening.Relative to the wild type,the degradation rates of the mutant n-hexadecane were all reduced with a range of 16.67%to 84.35%.Furthermore,the results of the insertion sites analysis showed that these inserted gene mutants were predicted to be a serine protease,a ferredoxin,a hypothetical protein,a ferredoxin reductase,an ABC transporter,an ATP-binding protein,and a cytochrome(CYP153).In addition to the putative protein,some genes had functions different from those traditionally identified,suggesting that it could have other functions.Hence,to the best of our knowledge,we were the first to successfully construct a mutant library of the Dietzia genus bacteria,providing an important method for studying the mechanisms of hydrocarbon degradation as well as the high salinity and alkalinity tolerance of Dietzia bacteria.

关 键 词：随机突变体文库 Dietzia属 筛库 耐盐 烷烃降解 

分 类 号：X172[环境科学与工程—环境科学]