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cbpD基因对溶藻弧菌毒力及相关生物学特性的影响

苏雯晓 邓益琴 臧树军 王茜 林梓阳 冯娟

苏雯晓, 邓益琴, 臧树军, 王茜, 林梓阳, 冯娟. cbpD基因对溶藻弧菌毒力及相关生物学特性的影响[J]. 南方水产科学. doi: 10.12131/20220025
引用本文: 苏雯晓, 邓益琴, 臧树军, 王茜, 林梓阳, 冯娟. cbpD基因对溶藻弧菌毒力及相关生物学特性的影响[J]. 南方水产科学. doi: 10.12131/20220025
SU Wenxiao, DENG Yiqin, ZANG Shujun, WANG Qian, LIN Ziyang, FENG Juan. Effects of cbpD gene on virulence and related biological characteristics of Vibrio alginolyticus[J]. South China Fisheries Science. doi: 10.12131/20220025
Citation: SU Wenxiao, DENG Yiqin, ZANG Shujun, WANG Qian, LIN Ziyang, FENG Juan. Effects of cbpD gene on virulence and related biological characteristics of Vibrio alginolyticus[J]. South China Fisheries Science. doi: 10.12131/20220025

cbpD基因对溶藻弧菌毒力及相关生物学特性的影响

doi: 10.12131/20220025
基金项目: 国家自然科学基金项目 (31902415);广东省自然科学基金项目 (2019A1515011833);中国水产科学研究院中央级公益性科研院所基本科研业务费专项资金资助 (2020XT0407)
详细信息
    作者简介:

    苏雯晓 (1996—),女,硕士研究生,研究方向为水产动物病原学。E-mail: sosmcx1989@qq.com

    通讯作者:

    邓益琴 (1990—),女,副研究员,博士,从事鱼类细菌病及防治技术研究。E-mail: yiqindd@126.com

    冯 娟 (1973—),女,研究员,博士,从事水产动物免疫与防治技术研究。E-mail: jannyfeng@163.com

  • 中图分类号: R917.1

Effects of cbpD gene on virulence and related biological characteristics of Vibrio alginolyticus

  • 摘要: 为研究几丁质结合蛋白 (the Chitin-binding protein D, CbpD) 对溶藻弧菌 (Vibrio alginolyticus) 毒力和相关生物学特性的影响,通过同源重组技术构建溶藻弧菌ZJ-T的cbpD基因缺失突变株ZJ-T-ΔcbpD,比较突变株与野生株对斑马鱼的毒性,以及毒力相关生理过程,包括生长能力、菌落形态、运动性、胞外蛋白酶分泌活性、溶血活性、抗生素敏感性、生物膜形成、过氧化氢和铜离子抗性以及铁离子吸收能力的差异。研究发现,cbpD缺失后,溶藻弧菌对斑马鱼的毒力显著减弱;cbpD缺失后,细菌游动能力、涌动能力和胞外蛋白酶活性均降低,突变株对铜离子Cu2+的抗性提高;cbpD的突变不影响溶藻弧菌在富营养培养基中的生长、生物膜的形成,溶血活性、对大部分抗生素敏感性、对过氧化氢 (H2O2) 的抗性和铁离子Fe3+的获取能力。结果表明,cbpD可能通过正调控溶藻弧菌的运动能力和胞外蛋白酶分泌活性,而促进溶藻弧菌的毒力。
  • 图  1  缺失株ZJ-T-ΔcbpD的构建

    Figure  1.  Construction of deletion strain ZJ-T-ΔcbpD

    图  2  LBS培养基中的生长曲线

    Figure  2.  Growth curves in LBS culture

    图  3  溶藻弧菌ZJ-T和突变株ZJ-T-ΔcbpD的运动性

    Figure  3.  Mobility of V. alginolyticus ZJ-T and mutant strain ZJ-T-ΔcbpD

    图  4  运动性统计分析

    Figure  4.  Motilitystatistical analysis

    图  5  蛋白酶分泌能力

    Figure  5.  Secretion of extracellular protease capacity

    图  6  溶藻弧菌ZJ-T和ZJ-T-ΔcbpD的溶血性

    Figure  6.  Hemolytic activity of V. alginolyticus ZJ-T and ZJ-T-ΔcbpD

    图  7  溶藻弧菌ZJ-T和ZJ-T-ΔcbpD对过氧化氢、硫酸铜以及铁离子螯合剂2-2'-联吡啶的敏感性

    Figure  7.  Sensitivity to H2O2, CuSO4 and DIP of wild type and cbpD mutant on LBS plate

    图  8  生物膜形成量

    Figure  8.  Biofilm formation

    图  9  溶藻弧菌ZJ-T和突变株ZJ-T-ΔcbpD感染斑马鱼后存活率

    Figure  9.  Survival rate of V. alginolyticus ZJ-T and mutant strain ZJ-T-ΔcbpD after infection of zebrafish

    表  1  本研究所用菌株及质粒

    Table  1.   Strains and plasmids used in this study

    菌株或质粒
    Strain or plasmid
    相关特征
    Relevant characteristic
    来源
    Source
    菌株 Strains
    Vibrio alginolyticus ZJ-T 氨苄抗性;野生菌株ZJ-51半透明/光滑变体;分离自中国南部沿海患病石斑鱼 [14]
    ZJ-T-ΔcbpD 缺失cbpD基因的ZJ-T突变株 本文构建
    E.coliⅡ3813 lacIQ,thi1,supE44,endA1,recA1,hsdR17,gyrA462,zei298::Tn10[Tc],ΔthyA::erm-pir116;自杀质粒pSW7848的中间宿主 [15]
    E.coli GEB883 大肠杆菌野生株K12 ΔdapA::ermpir RP4-2 ΔrecAgyrA462,zei298::Tn10;接合作用供体菌 [16]
    质粒 Plasmids
    pSW7848 氯霉素抗性;具有R6K起点的自杀质粒,需要pir蛋白进行复制,带ccdB毒性基因 [17]
    pSW7848-cbpDup-cbpDdown 氯霉素抗性;包括cbpD上下游同源臂的pSW7848质粒 本文构建
    下载: 导出CSV

    表  2  本研究所用引物

    Table  2.   Primers used in this study

    引物
    Primer
    序列 (5'—3')
    Sequence
    用途
    Application
    pSW7848_fwd GTCTGATTCGTTACCAATTATGACAAC 扩增pSW7848片段
    pSW7848_rev GAATTCGATATCAAGCTTATCGATAC
    cbpD-U-F ataagcttgatatcgaattcCAACATATGTGGGATAGTGCGT 扩增cbpD上游同源臂
    cbpD-U-R ggtccattctttaccCTGAAACACAAGCGGTTTAAGC
    cbpD-D-F aaccgcttgtgtttcagGGTAAAGAATGGACCGCTC 扩增cbpD下游同源臂
    cbpD-D-R taattggtaacgaatcagacGTCTTGGTTCTTAAAGGAGCTG
    Del-check-pSW7848-F TCACTGTCCCTTATTCGCACC 验证cbpD上下游片段是否与
    质粒pSW7848重组成功
    Del-check-pSW7848-R CTGCTTTTGAGCACTACCCG
    Del-cbpD-check-F GTCACAACATTACGGGATCTAAC 检验cbpD基因是否成功敲除
    Del-cbpD-check-R GCGAATAACGATATGTGCTCTG
    下载: 导出CSV

    表  3  药敏试验结果

    Table  3.   Drug sensitivity tests results of ZJ-T and ZJ-T-ΔcbpD

    药物   
    Drug   
    每片含量
    Content of each tablet/(μg·片−1)
    ZJ-TZJ-T-ΔcbpD
    抑菌圈直径
    Size of inhibition zone/mm
    敏感性
    Sensitivity
    抑菌圈直径
    Size of inhibition zone/mm
    敏感性
    Sensitivity
    克拉霉素 Claricid 15 18.24±1.10 S 15.59±0.81 I
    利福平 Rifampin 5 15.35±0.35 R 13.66±0.33 R
    阿莫西林 Amoxicillin 20 7.67±0.45 R 0 R
    多西环素 Doxycycline 30 12.79±0.32 I 13.08±0.19 I
    磺胺异噁唑 Sulfafurazole 300 0 R 0 R
    麦迪霉素 Medimycin 30 9.63±0.64 R 8.52±0.12 R
    头孢氨苄 Cephalexin 30 8.06±0.62 R 7.40±0.24 R
    氟苯尼考 Florfenicol 30 22.15±1.31 S 20.96±0.78 S
    妥布霉素 Tobramycin 10 15.07±1.32 S 15.25±2.01 S
    环丙沙星 Ciprofloxacin 5 16.37±1.07 I 16.98±1.56 I
    恩诺沙星 Enrofloxacin 10 15.04±0.87 R 14.91±0.44 R
    呋喃唑酮 Furazolidone 100 11.77±1.08 R 10.81±0.43 R
    复方新诺明(SMZ/TMP) 23.75/1.25 0 R 0 R
    庆大霉素 Gentamicin 10 13.43±0.47 I 12.38±0.17 I
    链霉素 Streptomycin 10 8.12±0.16 R 8.08±0.12 R
    头孢唑啉 Cefazolin 30 10.01±0.78 R 9.73±0.04 R
    四环素 Tetracycline 30 12.89±0.73 R 12.90±1.15 R
    红霉素 Erythromycin 15 16.86±0.91 I 15.04±0.68 I
    氯霉素 Chloramphenicol 30 20.93±1.08 S 21.19±0.45 S
    氨苄西林 Ampicillin 10 0 R 0 R
    氧氟沙星 Ofloxacin 5 15.72±0.73 I 15.88±0.28 I
    诺氟沙星 Norfloxacin 10 14.24±0.62 I 14.75±0.84 I
    万古霉素 Vancomycin 30 0 R 0 R
    注:R. 耐药;I. 中度敏感;S. 高度敏感。 Note: R. Resistance; I. Sensitivity; S. Highly sensitivity.
    下载: 导出CSV
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出版历程
  • 收稿日期:  2022-01-24
  • 修回日期:  2022-03-01
  • 录用日期:  2022-03-11
  • 网络出版日期:  2022-03-26

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