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
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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 B1 H 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 c DNA library. B1 H system can be successfully applied in screening the DNA binding proteins in methylotrophic bacteria. And the establishment of this method laid a solidfoundation to clarify the regulatory mechanism of methanol dehydrogenase gene in the process of development and metabolic product in the methylotrophic bacteria.
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 B1 H 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 c DNA library. B1 H system can be successfully applied in screening the DNA binding proteins in methylotrophic bacteria. And the establishment of this method laid a solidfoundation to clarify the regulatory mechanism of methanol dehydrogenase gene in the process of development and metabolic product in the methylotrophic bacteria.
引文
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[15] ANDREEVA I G,GOLUBEVA L I,KUVAEVA T M,et al.Identification of Pantoea ananatis gene encoding membrane pyrroloquinoline quinone(PQQ)-dependent glucose dehydrogenase and pqq ABCDEF operon essential for PQQ biosynthesis[J]. FEMS Microbiology Letters,2011,318(1):55-60.
[16] WANG H C,KO T P,WU M L,et al. Neisseriaconserved protein DMP19 is a DNA mimic protein that prevents DNA binding to a hypothetical nitrogen-response transcription factor[J]. Nucleic Acids Research,2012,40(12):5718-5730.
[17]萨姆布鲁克J,拉塞尔D W.分子克隆实验指南[M]. 3版.北京:科学出版社,2016.
[2]唐靓,张岭,李林子,等.吡咯喹啉醌研究新进展[J].食品科学,2005,19(2):287-291.TANG L,ZHANG L,LI L Z,et al. Lastest progress in research on pyrroloquinoline quinone[J]. Food Science,2005,19(2):287-291.(in Chinese with English abstract)
[3]冷春玲,宋洁.吡咯喹啉醌生物学功能研究进展[J].辽东学院学报(自然科学版),2014,21(2):103-108.LENG C L,SONG J. Biological function of pyrroloquinoline quinine:research progress[J]. Journal of Liaodong University(Natural Sciences),2014,21(2):103-108.(in Chinese with English abstract)
[4]周怡雯,陈建华.新辅酶吡咯喹啉醌研究进展[J].中国生化药物杂志,2008,29(4):279-282.ZHOU Y W,CHEN J H. Progress in the research of pyrroloquinoline quinone[J]. Chinese Journal of Biochemical Pharmaceutics,2008,29(4):279-282.(in Chinese with English abstract)
[5] LI J,GAN J H,MATHEWS F S,et al. The enzymatic reaction-induced configuration change of the prosthetic group PQQ of methanol dehydrogenase[J]. Biochemical and Biophysical Research Communications,2011,406(4):621-626.
[6]李大攀,葛欣,魏静远,等.甲基营养菌MP688甲醇脱氢酶基因mpq1818的敲除及功能研究[J].生物技术通讯,2015,25(5):632-635.LI D P,GE X,WEI J Y,et al. Knockout and characterization of mpq1818 gene of Methylovorus sp. MP688[J]. Letters in Biotechnology,2015,25(5):632-635.(in Chinese with English abstract)
[7]王冠芳,王银善,徐宁,等.依赖PQQ甲醇脱氢酶对底物催化专一性差的原因分析[J].微生物学杂志,2002,22(1):4-6.WANG G F,WANG Y S,XU N,et al. Cause analysis of poor substrate catalytic specificity of PQQ-dependent methanol dehydrogenase[J]. Journal of Microbilogy,2002,22(1):4-6.(in Chinese with English abstract)
[8]李淼鑫,熊向华,汪建华,等.甲基营养菌甲醇脱氢酶基因的克隆与表达[J].生物技术通讯,2010,21(6):779-782.LI M X,XIONG X H,WANG J H,et al. Cloning and expression of methanol dehydrogenase gene of a methylotrophic bacteria strain[J]. Letters in Biotechnology,2010,21(6):779-782.(in Chinese with English abstract)
[9] XIONG X H,ZHI J J,YANG L,et al. Complete genome sequence of the bacterium Methylovorus sp. strain MP688,a high-level producer of pyrroloquinolone quinone[J]. Journal of Bacteriology,2011,193(4):1012-1013.
[10] MENG X,BRODSKY M H,WOLFE A S A. A bacterial one-hybrid system for determining the DNA-binding specificity of transcription factors[J]. Nature Biotechnology,2005,23(8):988-994.
[11] DUARI S,BOSLEY A,ABULENCIA A B,et al. A bacterial one-hybrid selection system for interrogating zinc fingerDNA interactions[J]. Combinational Chemistry&High Throughput Screening,2006,9(4):301-311.
[12]廖名湘,方福德.酵母单杂交体系:一种研究DNA-蛋白质相互作用的有效方法[J].中国医学科学院学报,2000,22(4):388-391.LIAO M X,FANG F D. Yeast one-hybrid system:one effective method studying DNA-protein interaction[J]. Acta Academiae Medicinae Sinicae,2000,22(4):388-391.(in Chinese with English abstract)
[13]马守东,洪源,成军.酵母单杂交技术的原理及应用[J].世界华人消化杂志,2003,11(4):450-451.MA S D,HONG Y,CHENG J. Principle and application of yeast single hybridization[J]. Word Chinese Journal of Digestology,2003,11(4):450-451.(in Chinese with English abstract)
[14] SHEN Y Q,BONNOT F,IMSAND E M,et al. Distribution and properties of the genes encoding the biosynthesis of the bacterial cofactor,pyrroloquinoline quinone[J]. Biochemistry,2012,51(11):2265-2275.
[15] ANDREEVA I G,GOLUBEVA L I,KUVAEVA T M,et al.Identification of Pantoea ananatis gene encoding membrane pyrroloquinoline quinone(PQQ)-dependent glucose dehydrogenase and pqq ABCDEF operon essential for PQQ biosynthesis[J]. FEMS Microbiology Letters,2011,318(1):55-60.
[16] WANG H C,KO T P,WU M L,et al. Neisseriaconserved protein DMP19 is a DNA mimic protein that prevents DNA binding to a hypothetical nitrogen-response transcription factor[J]. Nucleic Acids Research,2012,40(12):5718-5730.
[17]萨姆布鲁克J,拉塞尔D W.分子克隆实验指南[M]. 3版.北京:科学出版社,2016.