水稻白叶枯病菌c-di-GMP受体Clpxoo关键结合功能位点的确定

生物技术通报 ›› 2016, Vol. 32 ›› Issue (12): 124-129.

水稻白叶枯病菌c-di-GMP受体Clpxoo关键结合功能位点的确定

李建宇，李波,陈华民,杨凤环,何晨阳,田芳

中国农业科学院植物保护研究所植物病虫害生物学国家重点实验室，北京 100193

收稿日期:2016-05-04 出版日期:2016-12-25 发布日期:2016-12-07
作者简介:李建宇，男，硕士，研究方向：分子植物病理学；E-mail：lijianyugood@126.com
基金资助:
国家自然科学基金项目（31100947），北京市自然科学基金项目（5142017）

Identification of Critical Residues in c-di-GMP Receptor Clpxoo from Xanthomonas oryzae pv. oryzae

LI Jian-yu,LI Bo,CHEN Hua-min,YANG Feng-huan,HE Chen-yang,TIAN Fang

State Key Laboratory for Biology of Plant Diseases and Insect Pests，Institute of Plant Protection，Chinese Academy of Agricultural Sciences，Beijing 100193

Received:2016-05-04 Published:2016-12-25 Online:2016-12-07

摘要/Abstract

摘要： 旨在确定水稻白叶枯病菌（Xanthomonas oryzae pv. oryzae，Xoo）c-di-GMP信号受体Clpxoo的关键功能位点。通过对Clpxoo蛋白氨基酸位点基因突变，表达载体构建、蛋白诱导表达及其Ni-NTA Resin亲和层析，进行了Clpxoo 及其点突变蛋白的原核表达和产物纯化；通过等温滴定量热法（ITC），检测了Clpxoo 及其点突变蛋白与c-di-GMP的结合作用。利用基因定点突变和桥接PCR方法，在优化的诱导表达和纯化条件下，成功地获得了Clpxoo点突变蛋白和与c-di-GMP不发生结合的Clpxoo点突变体。结果表明，Clpxoo蛋白第70位天冬氨酸和第99位谷氨酸是与c-di-GMP结合的关键功能位点。

关键词: 水稻白叶枯病菌, c-di-GMP, 信号受体蛋白, 点突变体, 结合作用

Abstract: The purpose of this study is to identify the critical residues in Clpxoo of a c-di-GMP signal receptor in Xanthomonas oryzae pv. oryzae（Xoo）. By the gene mutation of amino acid site of Clpxoo protein，the construction of expressing vector，induced protein expressed，and analysis of Ni-NTA Resin affinity chromatography，the prokaryotic expression of Clpxoo and point mutants was conducted and the protein was purified.. The binding affinities of c-di-GMP with native and variant Clpxoo were measured by isothermal titration calorimetry（ITC）experiments. Under optimized conditions for protein expression and purification，the proteins of Clpxoo point mutants and point mutants of Clpxoo that does not bind with c-di-GMP were acquired successfully while using gene site-directed mutagenesis and bridging PCR. Results showed that site 70 of aspartic acids and site 99 of glutamic acid were found to be the key residues for their binding to c-di-GMP.

Key words: Xanthomonas oryzae pv. pryzae, c-di-GMP, signal receptor protein, point mutant, binding affinity

中图分类号:

10.13560/j.cnki.biotech.bull.1985.2016.12.020

李建宇，李波,陈华民,杨凤环,何晨阳,田芳. 水稻白叶枯病菌c-di-GMP受体Clpxoo关键结合功能位点的确定[J]. 生物技术通报, 2016, 32(12): 124-129.

LI Jian-yu,LI Bo,CHEN Hua-min,YANG Feng-huan,HE Chen-yang,TIAN Fang. Identification of Critical Residues in c-di-GMP Receptor Clpxoo from Xanthomonas oryzae pv. oryzae[J]. Biotechnology Bulletin, 2016, 32(12): 124-129.

参考文献

[1] He YW, Xu M, Lin K, et al. Genome scale analysis of diffusible signal factor regulon in Xanthomonas campestris pv. campestris：identification of novel cell&ndash；cell communication dependent genes and functions[J] . Gastrointestinal Endoscopy, 2000, 51（1）：73-76.
[2] 管文静, 吴茂森, 何晨阳. 水稻白叶枯病菌核苷酸信号受体蛋白Clpxoo的分子鉴定及其功能[J] . 微生物学报, 2009, 49（1）：32-37.
[3] He YW, Ng Alvin YJ, Xu M, et al. Xanthomonas campestris cell-cell communication involves a putative nucleotide receptor protein Clp and a hierarchical signaling network[J] . Molecular Microbiology, 2007, 64（2）：281-292.
[4] Hsiao YM, Liao HY, Lee MC, et al. Clp upregulates transcription of engA gene encoding a virulence factor in Xanthomonas campestris by direct binding to the upstream tandem Clp sites[J] . Febs Letters, 2005, 579（17）：3525-3533.
[5] Jonas K, Melefors O, R?mling U. Regulation of c-di-GMP metabolism in biofilms[J] . Future Microbiology, 2009, 4（3）：341-358.
[6] Regine H. Principles of c-di-GMP signalling in bacteria[J] . Nature Reviews Microbiology, 2009, 7（7）：263-273.
[7] Kalia D, Merey G, Nakayama S, et al. ChemInform abstract：Nucleotide, c-di-GMP, c-di-AMP, cGMP, cAMP, （p）ppGpp signaling in bacteria and implications in pathogenesis[J] . Chemical Society Reviews, 2012, 42（1）：305-341.
[8] R?mling U, Galperin MY, Gomelsky M. Cyclic di-GMP：the First 25 Years of a Universal Bacterial Second Messenger[J] . Microbiology & Molecular Biology Reviews Mmbr, 2013, 77（1）：1-52.
[9] Krasteva PV, Fong JC, Shikuma NJ. Vibrio cholerae VpsT regulates matrix production and motility by directly sensing cyclic di-GMP[J] . Science, 2010, 327（5967）：866-868.
[10] Newell PD, Monds RD, O’Toole GA. LapD is a bis-（3’, 5’）-cyclic dimeric GMP-binding protein that regulates surface attachment by Pseudomonas fluorescens Pf0-1[J] . Proceedings of the National Academy of Sciences of the United States of America, 2009, 106（9）：3461-3466.
[11] Sondermann H, Shikuma NJ, Yildiz FH. You’ve come a long way：c-di-GMP signaling[J] . Current Opinion in Microbiology, 2012, 15（2）：140-146.
[12] Chin KH, Lee YZ. The cAMP receptor-like protein CLP is a novel c-di-GMP receptor linking cell-cell signaling to virulence gene expression in Xanthomonas campestris[J] . Journal of Molecular Biology, 2007, 396（3）：646-662.
[13] Tao F, He YW, Wang DH, et al. The cyclic nucleotide monophosphate domain of Xanthomonas campestris global regulator Clp defines a new class of cyclic di-GMP effectors[J] . Journal of Bacteriology, 2010, 192（4）：1020-1029.
[14] 戴灿, 苗聪秀, 卢光琇. 基于重叠延伸PCR法的定点突变技术[J] . 现代生物医学进展, 2010, 10（3）：411-412.
[15] 闫振文, 梁秀龄, 杨春水, 等. PCR技术对Wilson病基因进行定点突变的研究[J] . 中山医科大学学报, 2002, 23（2）：94-96.
[16] Yang FH, Tian F, Sun L, et al. A novel two-component system PdeK/PdeR regulates c-di-GMP turnover and virulence of Xanthomonas oryzae pv. oryzae[J] . Molecular Plant-Microbe Interactions, 2012, 25（10）：1361-1369.
[17] Massimo M, Lee VT, Mamoru H, et al. The second messenger bis-（3’-5’）-cyclic-GMP and its PilZ domain-containing receptor Alg44 are required for alginate biosynthesis in Pseudomonas aeruginosa[J] . Molecular Microbiology, 2007, 65（4）：876-895.
[18] Chen CL, Guo YF, Chen XW, et al. Efficient expression of Cap gene of porcine circovirus type 2 by pCold-SUMO expression vector[J] . Journal of South China Agricultural University, 2012, 33（3）：393-397.
[19] 李波, 田芳, 陈华民, 等. C-di-GMP抑制转录调控因子Clpxoo与葡聚糖酶基因启动子的结合[J] . 微生物学报, 2013, 53（11）：1166-1171.
[20] 胡晓环, 童威, 邵爽, 等. 等温滴定量热法研究牛血清白蛋白与十二烷基二羟乙基甲基溴化铵的相互作用[J] . 浙江大学学报：理学版, 2011, 38（5）：525-529.
[21] Baraquet C, Harwood CS. A FleQ DNA binding consensus sequence revealed by studies of FleQ-dependent regulation of biofilm gene expression in Pseudomonas aeruginosa[J] . Journal of Bacteriology, 2015, 198（1）：178-186.
[22] Baraquet C, Harwood CS. The FleQ protein from Pseudomonas aeruginosa functions as both a repressor and an activator to control gene expression from the pel operon promoter in response to c-di-GMP[J] . Nucleic Acids Research, 2012, 40（15）：7207-7218.
[23] Lu XH, An SQ, Tang DJ, et al. RsmA regulates biofilm formation in Xanthomonas campestris through a regulatory network involving cyclic di-GMP and the Clp transcription factor[J] . PLoS One, 2012, 7（12）：e52646.
[24] Kariisa AT, Grube A, Tamayo R, et al. Two nucleotide second messengers regulate the production of the Vibrio cholerae colonization factor GbpA[J] . BMC Microbiology, 2015, 15（1）：1-15.