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罗非鱼无乳链球菌ΔessA、ΔessB、ΔessC敲除菌株的构建及其特性分析

马艳平 郝乐 冯国清 梁志凌 马江耀 柯浩 刘振兴

马艳平, 郝乐, 冯国清, 梁志凌, 马江耀, 柯浩, 刘振兴. 罗非鱼无乳链球菌ΔessA、ΔessB、ΔessC敲除菌株的构建及其特性分析[J]. 南方水产科学. doi: 10.12131/20210246
引用本文: 马艳平, 郝乐, 冯国清, 梁志凌, 马江耀, 柯浩, 刘振兴. 罗非鱼无乳链球菌ΔessA、ΔessB、ΔessC敲除菌株的构建及其特性分析[J]. 南方水产科学. doi: 10.12131/20210246
MA Yanping, HAO Le, FENG Guoqing, LIANG Zhiling, MA Jiangyao, KE Hao, LIU Zhenxing. Construction and characterization research of essA, essB and essC-deleted mutants of Tilapia Streptococcus agalactiae[J]. South China Fisheries Science. doi: 10.12131/20210246
Citation: MA Yanping, HAO Le, FENG Guoqing, LIANG Zhiling, MA Jiangyao, KE Hao, LIU Zhenxing. Construction and characterization research of essA, essB and essC-deleted mutants of Tilapia Streptococcus agalactiae[J]. South China Fisheries Science. doi: 10.12131/20210246

罗非鱼无乳链球菌ΔessA、ΔessB、ΔessC敲除菌株的构建及其特性分析

doi: 10.12131/20210246
基金项目: 广东省自然科学基金项目 (2021A1515010498);广东省农业科学院水产研究中心课题 (XT202232);佛山市财政专项资金—2021年度共建广东农业科技示范市项目
详细信息
    作者简介:

    马艳平  (1984—),女,副研究员,博士,从事水产病害与防控研究。 E-mail: mayanping@gdaas.cn

    通讯作者:

    刘振兴 (1981—),男,副研究员,博士,从事水产病害与防控研究。 E-mail: liuzhenxing@gdaas.cn

  • 中图分类号: S 943.116.42

Construction and characterization research of essA, essB and essC-deleted mutants of Tilapia Streptococcus agalactiae

  • 摘要: VII型分泌系统存在于无乳链球菌 (Streptococcus agalactiae) 中,其组成膜蛋白对底物蛋白的分泌转运至关重要。为此利用热敏型自杀质粒,构建了含氯霉素筛选标签的膜蛋白重组敲除载体pSET4s-ΔessA、pSET4s-ΔessB、pSET4s-ΔessC,经同源重组后,利用PCR技术筛选到了其缺失突变株-无乳链球菌ΔessA、ΔessB、ΔessC。通过菌株生长曲线分析,ΔessA、ΔessB、ΔessC敲除株均较野生株 (WT) 株生长过程变慢,其中ΔessA、ΔessB敲除株在生长早期有显著性差异 (P<0.01)。对ESAT6蛋白分泌影响分析显示,与WT株相比,ΔessA、ΔessB、ΔessC敲除株ESAT6的表达量均显著下调 (P<0.01)。攻毒试验结果显示,ΔessA、ΔessB、ΔessC敲除株均较WT株毒力显著降低 (P<0.01)。研究表明,essA、essB、essC为无乳链球菌VII型分泌系统重要的膜蛋白,其基因缺失造成了分泌蛋白ESAT6 mRNA表达水平下降,影响了菌株毒力。
  • 图  1  essAessBessC同源臂融合PCR结果

    Figure  1.  Overlap extension PCR results of essA, essB, essC homologous arms

    图  2  敲除株ΔessA、ΔessB、ΔessC DNA水平PCR鉴定结果

    Figure  2.  PCR identification results of ΔessA, ΔessB, ΔessC knockout strains at DNA level

    图  3  敲除株ΔessA、ΔessB、ΔessC RNA水平PCR鉴定结果

    Figure  3.  PCR identification of ΔessA, ΔessB, ΔessC knockout strains at RNA level

    图  4  敲除菌株ΔessA、ΔessB、ΔessC 生长曲线测定结果

    Figure  4.  Growth curve of ΔessA, ΔessB and ΔessC knockout strains

    图  5  ESAT6 qPCR检测方法的建立

    Figure  5.  qPCR detection method of ESAT6 gene

    图  6  敲除株ΔessA、ΔessB、ΔessC 与野生株对底物蛋白ESAT6表达量的影响结果

    Figure  6.  Effect of substrate protein ESAT6 expression by knockout strains ΔessA, ΔessB, ΔessC and WT strain

    图  7  敲除菌株ΔessA、ΔessB、ΔessC 与野生株毒力试验结果

    Figure  7.  Virulence test of ΔessA, ΔessB, ΔessC knockout strains and WT strain

    图  8  攻毒死亡鱼解剖及其菌株鉴定结果

    Figure  8.  Anatomical symptom result and identification of strains of infected dead tilapia

    表  1  试验用引物

    Table  1.   Primers in this study

    引物名称 Primer name引物序列5'—3' Primers sequence 5'—3'
    重组质粒构建用引物 Primers for recombinant plasmid construction
    essAup-FBamHI 5'-GCGGATCCGCCGTAGGCACAGTAGCGACT-3' BamHI
    essAup-R 5'-GAGTGAGACTTTAGATTGACGG-3'
    essAdown-F 5'-ACGTTGAGCCTCGGAACCCATCGAATTAGAATACACATATTAACATTACT-3'
    essAdown-REcoRI 5'-GCGAATTCGATAGAGCGTGCGCTTCTGTCTG-3' EcoRI
    essBup-FBamHI 5'-GCGGATCCGGACCAGATTTTTGGCAGTTATC-3' BamHI
    essBup-R 5'-CAGCCTGAATAGTTTCTGCATGAT-3'
    essBdown-F 5'-GACGTTGAGCCTCGGAACCCATCGAATTATAAAGTTGATTATAATCAAGTGA-3'
    essBdown-REcoRI 5'-GCGAATTCAGAAGATGTACTTGTTGTAGTAC-3' EcoRI
    essCup-FSalI 5'-GCGTCGACAAACGGCAAGTCGCCAATACCAG-3'SalI
    essCup-R 5'-TGCTGACTCCAATCATCACTAG-3'
    essCdown-F 5'-GACGTTGAGCCTCGGAACCCATCGAATTACAGGAAATGGCTGATACTTATCAC-3'
    essCdown-REcoRI 5'-GCGAATTCTTAACTTTTCATCAGTTACAAT-3' EcoRI
    Cat(essA)-F 5'-ATGAAAACCGTCAATCTAAAGTCTCACTCCACCGAACTAGAGCTTGATG-3'
    Cat(essB)-F 5'-TATCATGCAGAAACTATTCAGGCTGCACCGAACTAGAGCTTGATGAAAA-3'
    Cat(essC)-F 5'-CCTCCTCTAGTGATGATTGGAGTCAGCACACCGAACTAGAGCTTGATGAA-3'
    Cat-R 5'-TAATTCGATGGGTTCCGAGGCTC-3'
    基因缺失突变株筛选所用引物 Primers for gene deletion mutant strains screening
    essA验证-F 5'-GCCGTAGGCACAGTAGCGACTG-3'
    essA验证-R 5'-ACATTCAAGGCTAATCGTAA-3'
    essB验证-F 5'-TGGAAACAGATTCGTTTGTA-3'
    essB验证-R 5'-TACCTACTTTTAGTTTTAG-3'
    essC验证-F 5'-CGTCCTCGTGGTATCTATATC-3'
    essC验证-R 5'-CAATATCATCCTGACCACGTAAG-3'
    essA-F 5'-ATGAAGTTGAAACGATTTTTAG-3'
    essA-R 5'-TTAATATGTGTATTCATCCT-3'
    essB-F 5'-TGGAAACAGATTCGTTTGTA-3'
    essB-R 5'-TACCTACTTTTAGTTTTAGA-3'
    essC-F 5'-TCGTGGTATCTATATCATTGCAAC-3'
    essC-R 5'-GATAAACAATATCATCCTGACCAC-3'
    Real-time PCR所用引物 Primers for real-time PCR
    ESAT6-F 5'-ATACTGCTGGTTCTCAACAA-3'
    ESAT6-R 5'-GTCAATAACTGCTTGCTCTT-3'
    16S-rRNA-F 5'-CGACGATACATAGCCGACC-3'
    16S-rRNA-R 5'-CCGTCACTTGGTAGATTTTCC-3'
    注:下划线为限制性内切酶位点。 Note: The underlined are restriction endonuclease sites.
    下载: 导出CSV
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  • 收稿日期:  2021-08-26
  • 修回日期:  2021-12-14
  • 录用日期:  2021-12-21
  • 网络出版日期:  2022-02-07

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