单增李斯特菌核糖核酸酶Rnase III RncS氨基酸突变对其降解RNA活性的影响

doi:10.13560/j.cnki.biotech.bull.1985.2018-0861

生物技术通报 ›› 2019, Vol. 35 ›› Issue (3): 110-116.doi: 10.13560/j.cnki.biotech.bull.1985.2018-0861

单增李斯特菌核糖核酸酶Rnase III RncS氨基酸突变对其降解RNA活性的影响

王立霞¹, 孟庆玲¹, 乔军¹, 蔡扩军², 王登峰³, 伍晔晖¹, 郭晶¹, 才学鹏⁴

1. 石河子大学动物科技学院,石河子 832003;
2. 乌鲁木齐市动物疾病控制与诊断中心,乌鲁木齐 830063;
3. 新疆畜牧科学院兽医研究所,乌鲁木齐 830000;
4. 中国农业科学院兰州兽医研究所,兰州 730046

收稿日期:2018-10-09 出版日期:2019-03-26 发布日期:2019-04-03
作者简介:王立霞,女,硕士研究生,研究方向：病原分子生物学;E-mail：645432907@qq.com
基金资助:
国家自然科学基金项目（31360596,30960274）,国家重点研发计划子课题（2016YFD0500900）,国家国际科技合作专项（2014DFR31310）,乌鲁木齐市科技局渝乌科技合作项目（Y161220001,14DFR31310）

Effects of Amino Acid Mutation in the RnaseIII RncS of Listeria monocytogenes on RNA Degradation Activity

WANG Li-xia¹, MENG Qing-ling¹, QIAO Jun¹, CAI Kuo-jun², WANG Deng-feng³, WU Ye-hui¹, GUO Jing¹, CAI Xue-peng⁴

1. College of Animal Science and Technology,Shihezi University,Shihezi 832003;
2. Animal Disease Control and Diagnosis Center in Urumqi,Urumqi 830063;
3. Institute of Veterinary Medicine,Xinjiang Academy of Animal Science,Urumqi 830000;
4. Lanzhou Veterinary Research Institute,Chinese Academy of Agricultural Sciences,Lanzhou 730046

Received:2018-10-09 Published:2019-03-26 Online:2019-04-03

摘要/Abstract

摘要： 为研究单增李斯特菌（LM）核糖核酸酶RnaseⅢ RncS氨基酸突变对RNA降解活性的影响。利用生物信息学软件分析单核细胞增生李斯特菌（LM）野毒株SB5中rncS基因编码的RnaseⅢ的结构域,并选择关键氨基酸利用基因重叠延伸PCR（SOE-PCR）技术对其进行了基因突变;然后将rncS突变基因片段D50A、E122A克隆至表达载体pET-32a（+）,在大肠杆菌中利用IPTG进行诱导表达;应用SDS-PAGE和Western Blot鉴定重组蛋白的表达情况及其抗原特异性;通过体外酶活试验研究其对RNA降解活性的影响。结构域分析结果显示,LM-RnaseⅢ氨基酸序列含有1个双链RNA结合结构域（DSRM）和1个核酸酶结构域（RIBOc）,其中结构域RIBOc含有5个活性位点。SDS-PAGE检测结果显示,表达的重组突变型RnaseⅢ-D50A和RnaseⅢ-E122 A蛋白相对分子质量均为42.5 kD,与理论值相符;Western blot分析表明重组突变型RnaseⅢ-D50A和RnaseⅢ-E122A蛋白可与LM阳性血清发生免疫学反应。体外酶活实验表明,RnaseⅢ发挥降解活性依赖于Mn²⁺或Mg²⁺,将其第50位天冬氨酸突变后,RnaseⅢ RncS的降解活性有所降低（P<0.001）;第122位谷氨酸突变后,RnaseⅢ RncS 降解活性极显著下降（P<0.0001）,提示第122位谷氨酸是维持LM RnaseⅢ RncS酶活性的关键位点。

关键词: 单核细胞增生李斯特菌, 核糖核酸酶RnaseⅢ, rncS基因, 降解活性

Abstract: To investigate the effect of amino acid mutation in Listeria monocytogenes（LM）ribonuclease RnaseIII RncS on RNA degradation activity,bioinformatics software was used to analyze the domains of RnaseIII encoded by rncS gene of LM wild-type strain SB5,and gene overlap extension PCR（SOE-PCR）was to perform its gene mutations. Then the rncS mutant gene fragments D50A and E122A were cloned into the expression vector pET-32a（+）,and induced by IPTG in Escherichia coli to generate mutant RnaseⅢ. SDS-PAGE and Western Blot were adapted to detect the expression form and the antigen specificity of fusion protein,respectively. Finally,the enzyme activity assay in vitro was to determine the effect of amino acid mutation on RNA degradation activity. The domain analysis revealed that the LM-RnaseIII protein amino acid sequence included a nuclease domain（RIBOc）,in which there were 5 active sites,and a double-stranded RNA binding domain（DSRM）. The results of SDS-PAGE showed that the relative molecular masses of the recombinant mutants RnaseIII-D50A and RnaseIII-E122 A were 42.5 kD,which was consistent with the theoretical values. Western Blot showed that the recombinant mutant RnaseIII-D50A and RnaseIII-E122A reacted with positive serum against LM. Enzyme activity experiments demonstrated that the degradation activity of RnaseIII was dependent on Mn²⁺ or Mg²⁺. After the 50th aspartic acid was mutated,the degradation activity of RnaseIII RncS reduced（P <0.001）,while it significantly（P <0.0001）decreased after the mutation of the 122nd glutamate,suggesting that the 122nd glutamate was the key site for maintaining the activity of LM RnaseIII. This study provides a scientific basis for further revealing the characteristics and mechanism of LM RncS.

Key words: Listeria monocytogenes, ribonuclease Rnase Ⅲ, rncS gene, degradation activity

王立霞, 孟庆玲, 乔军, 蔡扩军, 王登峰, 伍晔晖, 郭晶, 才学鹏. 单增李斯特菌核糖核酸酶Rnase III RncS氨基酸突变对其降解RNA活性的影响[J]. 生物技术通报, 2019, 35(3): 110-116.

WANG Li-xia, MENG Qing-ling, QIAO Jun, CAI Kuo-jun, WANG Deng-feng, WU Ye-hui, GUO Jing, CAI Xue-peng. Effects of Amino Acid Mutation in the RnaseIII RncS of Listeria monocytogenes on RNA Degradation Activity[J]. Biotechnology Bulletin, 2019, 35(3): 110-116.

参考文献

[1] Radoshevich L, Cossart P.Listeria monocytogenes:towards a complete picture of its physiology and pathogenesis.[J]. Nature Reviews Microbiology, 2018, 16(1):32.
[2] Gandhi M, Chikindas ML.Listeria:A foodborne pathogen that knows how to survive.[J]. International Journal of Food Microbiology, 2007, 113(1):1-15.
[3] 孙静娟, 邱景璇, 曾海娟, 等. 单增李斯特菌CdaA的抗原表位分析及抗体的制备[J]. 生物技术通报, 2018, 34(5):163-171.
[4] Neuhaus K, Satorhelyi P, Schauer K, et al.Acid shock of Listeria monocytogenes at low environmental temperatures induces prfA, epithelial cell invasion, and lethality towards Caenorhabditis elegans[J]. BMC Genomics, 2013, 27(14):285.
[5] Galié S, Garcíagutiérrez C, Miguélez EM, et al.Biofilms in the food industry:Health aspects and control methods[J]. Frontiers in Microbiology, 2018, 9:898.
[6] Van d VS, Van SS, Molenaar D, et al. The SOS response of Listeria
monocytogenes is involved in stress resistance and mutagenesis.[J]. Microbiology, 2010, 156(2):374-384.
[7] Grubaugh D, Regeimbal JR, Ghosh P, et al. The VirAB ABC transporter is required for VirR regulation of Listeria monocytogenes virulence and resistance to nisin[J]. Infection & Immol/Lunity, 2018, 86(3):IAI. 00901-17.
[8] 张再超, 彭叶龙, 孟庆玲, 等. 单核细胞增生李斯特菌ncRNA研究进展[J]. 生命的化学, 2013, 53(4):87-91.
[9] Arraiano CM, Andrade JM, Domingues S, et al.The critical role of RNA processing and degradation in the control of gene expression[J]. Fems Microbiology Reviews, 2010, 34(5):883-923.
[10] Afonyushkin T, Večerek B, Moll I, et al.Both RNase E and RNase III control the stability of sodB mRNA upon translational inhibition by the small regulatory RNA RyhB[J]. Nucleic Acids Research, 2005, 33(5):1678-1689.
[11] 王立霞, 孟庆玲, 蔡扩军, 等. 单核细胞增生李斯特菌核糖核酸酶RNaseⅢ RncS的表达及其生物学活性研究[J]. 中国预防兽医学报, 2018(7):581-585.
[12] Wagner EG, Altuvia S, Romby P.Antisense RNAs in bacteria and their genetic elements[J]. Advances in Genetics, 2002, 46(3):361-398.
[13] Darfeuille F, Unoson C, Vogel J, et al.An antisense RNA inhibits translation by competing with standby ribosomes[J]. Molecular Cell, 2007, 26(3):381-392.
[14] Lebreton A, Cossart P.RNA-and protein-mediated control of Listeria monocytogenes virulence gene expression[J]. RNA Biol. 2017, 14(5):460-470.
[15] Xiao J, Feehery CE, Tzertzinis G, et al.E. coli RNase III(E38A)generates discrete-sized products from long dsRNA[J]. RNA, 2009, 15(5):984-991.
[16] Blaszczyk J, Tropea JE, Bubunenko M, et al.Crystallographic and modeling studies of RNase III suggest a mechanism for double-stranded RNA cleavage[J]. Structure, 2001, 9(12):1225-1236.
[17] Sun W, Nicholson AW.Mechanism of action of Escherichia coli ribonuclease III. Stringent chemical requirement for the glutamic acid 117 side chain and Mn²+ rescue of the Glu117Asp mutant[J]. Biochemistry, 2001, 40(16):5102-5110.
[18] Gan J, Tropea JE, Austin BP, et al.Structural insight into the mechanism of double-stranded RNA processing by ribonuclease III[J]. Cell, 2006, 124(2):355-366.
[19] Robertson HD, Webster RE, Zinder ND.A nuclease specific for double-stranded RNA[J]. Virology, 1967, 32(4):718-719.
[20] Blaszczyk J, Gan J, Tropea JE, et al.Noncatalytic assembly of ribonuclease III with double-stranded RNA[J]. Structure, 2004, 12(3):457-466.
[21] Lioliou E, Sharma CM, Caldelari I, et al.Global regulatory functions of the, Staphylococcus aureus, endoribonuclease III in gene expression[J]. PLoS Genetics, 2012, 8(6):3202-3212.
[22] 许先进. 布鲁氏菌核糖核酸酶RibonucleaseⅢ的酶活性研究[D]. 武汉:华中农业大学, 2014.

单增李斯特菌核糖核酸酶Rnase III RncS氨基酸突变对其降解RNA活性的影响

Effects of Amino Acid Mutation in the RnaseIII RncS of Listeria monocytogenes on RNA Degradation Activity

PDF (PC)

可视化

摘要/Abstract

引用本文

使用本文

参考文献

相关文章 7

编辑推荐

Metrics

本文评价

[1]	杜冬冬, 钱晶, 李思琪, 刘雯菲, 魏向利, 刘长勇, 罗瑞峰, 康立超. 单核细胞增生李斯特菌LMXJ15全基因组测序及分析[J]. 生物技术通报, 2023, 39(7): 298-306.
[2]	任思雨, 姜聪一, 于涛, 康瑞, 姜晓冰. agr系统在单核增生细胞李斯特菌耐药及生物被膜形成中的作用[J]. 生物技术通报, 2023, 39(2): 254-262.
[3]	徐雅梦, 姜晓冰, 于涛. 单核细胞增生李斯特菌LadR蛋白对外排泵MdrL的调控机制研究[J]. 生物技术通报, 2018, 34(12): 166-171.
[4]	姜晓冰, 于涛, 牛亚冰, 徐雅梦, 石磊, 王海磊. 单核细胞增生李斯特菌喹诺酮耐药机制研究[J]. 生物技术通报, 2016, 32(7): 234-241.
[5]	王莉;冯飞飞;张强;冯莹颖;邓灵福;张晓莉;罗勤;. 单核细胞增生李斯特菌毒力基因inlB/actA双缺失突变株的构建[J]. , 2010, 0(11): 182-185.
[6]	冯莹颖;张晓莉;张强;罗勤;蒋苹;钱悦;冯爱平;. GFP用于单核细胞增生李斯特菌PrfA调控毒力基因actA转录表达的研究[J]. , 2009, 0(12): 139-143.
[7]	. 环境保护上的应用[J]. , 1991, 0(08): 112-117.