Biotechnology Bulletin ›› 2019, Vol. 35 ›› Issue (3): 110-116.doi: 10.13560/j.cnki.biotech.bull.1985.2018-0861

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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. 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 Online:2019-03-26 Published:2019-04-03

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