细菌单杂交方法在筛选甲基营养菌甲醇脱氢酶启动子结合蛋白中的应用  

Application of bacterial-one-hybrid( B1H) system in screening factors interacting with methanol dehydrogenase gene promoter in a methylotrophic bacteria strain

作　　者：邹琪琪 齐姗姗 谢录翰 辛琪 李俊乐[1] 张梦宁 葛欣[1] ZOU Qiqi;QI Shanshan;XIE Luhan;XIN Qi;LI Junyue;ZHANG Mengning;GE Xin(College of Life Science,Hebei University,Baoding 071002,China)

机构地区：[1]河北大学生命科学学院,河北保定071002

出　　处：《浙江农业学报》2018年第10期1705-1714,共10页Acta Agriculturae Zhejiangensis

基　　金：河北省自然科学基金(C2015201157;C2015201156);河北省高等学校科学技术研究项目(BJ2016008);河北大学实验室开放项目(sy201659)

摘　　要：为了构建甲基营养菌转录因子亚文库及筛选与甲醇脱氢酶启动子DNA相互作用的蛋白质,对甲基营养菌MP688的基因组和转录组数据进行分析,使用关键词对全基因注释信息和转录组结果进行搜索,找到相关基因的核苷酸序列,通过PCR获得目的片段并构建一系列转录因子亚文库,利用细菌单杂交系统筛选转录因子亚文库找到与甲醇脱氢酶启动子具有相互作用的调控因子。成功构建完成含有32个转录因子的亚文库,建立了甲基营养菌中细菌单杂交筛选方法,通过该方法筛选出2个与甲醇脱氢酶启动子具有强相互作用的转录因子基因。对于基因组已测序的细菌来说,构建转录因子亚文库筛选效率要优于构建基因组文库和c DNA文库,细菌单杂交系统能成功用于甲基营养菌,并能快速高效地筛选与启动子具有相互作用的转录因子。这一方法的建立,为阐明甲醇脱氢酶在甲基菌生长和代谢产物合成中的调控机制奠定了基础。To construct the transcription factor sublibrary of methylotrophic bacteria and screen the proteins interacting with the methanol dehydrogenase promoter,we found many candidate genes by analyzing the genome and transcriptome databases of methylotrophic bacteria(MP688)in the use of some key words searching the genome annotation and transcriptome result,then amplified the target genes by PCR(polymerase chain reaction)and construct a series of transcription factor sublibrary.Finally B1H and EMSA(electrophoretic mobility shift assay)methods were employed to screen and verify the candidate factors.We had constructed a total of 32 transcriptional factor clones,and two transcriptional factors which have strong interaction with methanol dehydrogenase promoter were screened out.For bacteria having detailed genomic sequence information,the construction of the transcriptional factor sublibrary is better than the construction of the genomic library or cDNA library.B1H system can be successfully applied in screening the DNA binding proteins in methylotrophic bacteria.And the establishment of this method laid a solid foundation to clarify the regulatory mechanism of methanol dehydrogenase gene in the process of development and metabolic product in the methylotrophic bacteria.

关 键 词：转录因子 文库构建 细菌单杂交系统 

分 类 号：Q935[生物学—微生物学]