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

doi:10.3969/j.issn.1004-1524.2018.10.14

浙江农业学报 ›› 2018, Vol. 30 ›› Issue (10): 1705-1714.DOI: 10.3969/j.issn.1004-1524.2018.10.14

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

邹琪琪, 齐姗姗, 谢录翰, 辛琪, 李俊乐, 张梦宁, 葛欣^*

河北大学生命科学学院,河北保定 071002

收稿日期:2018-01-15 出版日期:2018-10-25 发布日期:2018-11-02
通讯作者: 葛欣,E-mail:gexin88888@sina.com
作者简介:邹琪琪(1989—),女,山东泰安人,硕士研究生,主要从事微生物遗传育种研究。E-mail:zouqiqisunny@163.com
基金资助:
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)

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

ZOU Qiqi, QI Shanshan, XIE Luhan, XIN Qi, LI Junyue, ZHANG Mengning, GE Xin^*

College of Life Science, Hebei University, Baoding 071002, China

Received:2018-01-15 Online:2018-10-25 Published:2018-11-02

摘要/Abstract

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cDNA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。

关键词: 转录因子, 文库构建, 细菌单杂交系统

Abstract: 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.

Key words: transcriptional factor, gene library construction, bacterial-one-hybrid system

中图分类号:

Q935

邹琪琪, 齐姗姗, 谢录翰, 辛琪, 李俊乐, 张梦宁, 葛欣. 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[J]. 浙江农业学报, 2018, 30(10): 1705-1714.

ZOU Qiqi, QI Shanshan, XIE Luhan, XIN Qi, LI Junyue, ZHANG Mengning, GE Xin. Application of bacterial-one-hybrid (B1H) system in screening factors interacting with methanol dehydrogenase gene promoter in a methylotrophic bacteria strain[J]. , 2018, 30(10): 1705-1714.

参考文献

[1] 晁红军,宋修鹏,孙继华,等.甲基营养菌的研究进展[J].微生物学通报,2009,36(11):324-330.
CHAO H J, SONG X P, SUN J H, et al.Advances in methylotrophy[J]. Microbiology, 2009, 36(11):324-330. (in Chinese with English abstract)
[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 finger-DNA 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 pqqABCDEF 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.

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

RichHTML

PDF (PC)

可视化

摘要/Abstract

引用本文

使用本文

参考文献

相关文章 15

编辑推荐

Metrics

本文评价

[1]	何佳琦, 翟莹, 张军, 邱爽, 李铭杨, 赵艳, 张梅娟, 马天意. 大豆转录因子GmDof1.5的克隆与非生物胁迫诱导表达[J]. 浙江农业学报, 2021, 33(1): 1-7.
[2]	邱文怡, 王诗雨, 李晓芳, 徐恒, 张华, 朱英, 王良超. MYB转录因子参与植物非生物胁迫响应与植物激素应答的研究进展[J]. 浙江农业学报, 2020, 32(7): 1317-1328.
[3]	张古文, 沈立, 郑华章, 刘娜, 冯志娟, 龚亚明. 锌指蛋白转录因子Di19参与调控大豆干旱响应的研究进展[J]. 浙江农业学报, 2020, 32(2): 373-382.
[4]	李君霞, 王春义, 丁宇涛, 代书桃, 朱灿灿, 宋迎辉, 秦娜, 陈宇翔. MYB转录因子在植物耐盐基因工程中的应用进展[J]. 浙江农业学报, 2020, 32(10): 1910-1920.
[5]	刘慧洁, 徐恒, 邱文怡, 李晓芳, 张华, 朱英, 李春寿, 王良超. 转录因子在植物生长发育及非生物逆境响应的作用[J]. 浙江农业学报, 2019, 31(7): 1205-1214.
[6]	张旭, 王小佳, 黎思辰, 董甜甜, 汪志辉. 柑橘果实粒化过程中木质素生物合成与调控研究进展[J]. 浙江农业学报, 2019, 31(12): 2131-2140.
[7]	张莘, 郭晓敏, 常国斌, 朱鹏飞, 徐璐, 仇玲玲, 张扬, 徐琪, 陈国宏. 鸡NLRC5启动子区转录调控序列的初步研究[J]. 浙江农业学报, 2017, 29(3): 395-400.
[8]	李冬月, 原文霞, 郑超, 王栩鸣, 周洁, 严成其, 陈剑平. bZIP转录因子在植物激素介导的抗病抗逆途径中的作用[J]. 浙江农业学报, 2017, 29(1): 168-175.
[9]	冯琛，王玲，汤浩茹*，肖婕. 草莓MYB类转录因子FaMYB5基因的克隆与表达分析[J]. 浙江农业学报, 2016, 28(8): 1351-.
[10]	肖姗姗，赵虎，孙叶芳，戴余有*. 小兰屿蝴蝶兰（Phalaenopsis equestris）NAC转录因子家族的全基因组序列鉴定及其进化分析[J]. 浙江农业学报, 2016, 28(7): 1156-.
[11]	裴翠明，张振亚，马进*. 南方型紫花苜蓿叶片盐胁迫应答转录因子鉴定与分析[J]. 浙江农业学报, 2016, 28(4): 550-.
[12]	瓮巧云, 赵彦敏, 张贺, 宋晋辉, 马海莲, 袁进成, 刘颖慧. 玉米ZmREM基因的克隆与逆境胁迫后表达分析[J]. 浙江农业学报, 2016, 28(11): 1822-1827.
[13]	翟莹, 张军, 赵艳, 高士童, 孙婉姝, 张闯, 任巍巍, 王秀文, 王玉书. 大豆5个新发现ERF基因的生物信息学及表达分析[J]. 浙江农业学报, 2016, 28(10): 1644-1649.
[14]	苗立祥1，荣宁宁1，2，张豫超1，杨肖芳1，张琴3，蒋桂华1，*. 草莓花青苷分子调控机理的初步研究[J]. 浙江农业学报, 2015, 27(8): 1373-.
[15]	李雁杰1，2，朱丹华1，冯小锋3，高莎3，杨清华1，董德坤1，*. 含抗草甘膦CP4\\|EPSPS基因和耐逆Trihelix类转录因子GmGT\\|2A基因的植物表达载体构建及转化验证[J]. 浙江农业学报, 2015, 27(2): 133-.