海假交替单胞菌（<i>Pseudoalteromonas marina</i>）<i>pilZ</i>基因缺失抑制厚壳贻贝附着变态

张驰; 王劲松; 杨金龙; 张俊波; 万荣; 梁箫

doi:10.12284/hyxb2022090

海假交替单胞菌（Pseudoalteromonas marina）pilZ基因缺失抑制厚壳贻贝附着变态

doi: 10.12284/hyxb2022090

张驰^{1, 2,},
王劲松^{1, 2},
杨金龙^{1, 2, 3},
张俊波^{4, 5},
万荣^{4, 5, 6},
梁箫^{1, 2, ,}

1.
上海海洋大学国家海洋生物科学国际联合研究中心，上海 201306
2.
上海市水产动物良种创制与绿色养殖协同创新中心，上海 201306
3.
南方海洋科学与工程广东省实验室（广州），广东广州 511458
4.
上海海洋大学海洋科学学院，上海 201306
5.
国家远洋渔业工程技术研究中心，上海 201306
6.
国家远洋渔业工程技术研究中心舟山分中心，浙江舟山 316014

基金项目: 国家自然科学基金（41876159，41476131）；国家重点研发计划（2019YFC0312104，2020YFD0900804）；南方海洋科学与工程广东省实验室（广州）人才团队引进重大专项（GML2019ZD0402）；上海市优秀学术带头人计划（20XD1421800）。

详细信息

作者简介:
张驰（1996-），男，江苏省徐州市人，从事海洋贝类分子生物学研究。E-mail：M190100015@st.shou.edu.cn

通讯作者:
梁箫（1983-），女，主要从事海洋贝类分子生物学研究。E-mail: x-liang@shou.edu.cn

中图分类号: S968.31；Q938.8
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出版历程
- 收稿日期: 2021-11-10
- 修回日期: 2021-12-27
- 刊出日期: 2022-04-14

Knockout of Pseudoalteromonas marina pilZ gene inhibited the settlement and metamorphosis of Mytilus coruscus

Zhang Chi^{1, 2
,},
Wang Jinsong^{1, 2},
Yang Jinlong^{1, 2, 3},
Zhang Junbo^{4, 5},
Wan Rong^{4, 5, 6},
Liang Xiao^{1, 2
, ,}

1.
International Research Center for Marine Biosciences, Ministry of Science and Technology, Shanghai Ocean University, Shanghai 201306, China
2.
Shanghai Collaborative Innovation Center for Cultivating Elite Breeds and Green-Culture of Aquaculture Animals, Shanghai 201306, China
3.
Southern Marine Science and Engineering Guangdong Laboratory (Guangzhou), Guangzhou 511458, China
4.
College of Marine Sciences, Shanghai Ocean University, Shanghai 201306, China
5.
National Engineering Research Center for Oceanic Fisheries, Shanghai 201306, China
6.
Zhoushan Branch of National Engineering Research Center for Oceanic Fisheries, Zhoushan 316014, China

摘要

摘要: 为探究海假交替单胞菌pilZ基因的缺失对生物被膜形成及突变菌生物被膜对厚壳贻贝幼虫附着变态的影响，本文通过同源重组构建pilZ基因缺失菌，分析了基因缺失菌生物被膜的细菌密度、膜厚、c-di-GMP水平和胞外产物含量等特性的变化及其对厚壳贻贝幼虫附着变态的调控作用。结果表明：与野生型菌株相比，pilZ基因缺失菌形成的生物被膜膜厚增加、细菌数量增多，胞外产物中β-多糖、蛋白含量减少，抑制了厚壳贻贝幼虫的附着变态（p<0.05）；而c-di-GMP水平、α-多糖和脂质含量无显著变化（p>0.05）。由此可见，海假交替单胞菌pilZ基因的缺失可调控细菌生物被膜形成和胞外产物包括β-多糖、蛋白质的含量，从而抑制厚壳贻贝幼虫的附着变态。
- pilZ /
- 生物被膜 /
- 厚壳贻贝 /
- 附着变态 /
- 胞外产物
Abstract: To investigate the effect of Pseudoalteromonas marina pilZ gene knockout on the biofilm formation and its influence on the settlement and metamorphosis of Mytilus coruscus larvae, ΔpilZ was constructed by homologous recombination, and the changes in bacterial density, biofilm thickness, c-di-GMP level and extracellular polymeric substances (EPS) of ΔpilZ bacteria biofilm were analyzed, as well as the regulation of the settlement and metamorphosis of M. coruscus larvae. The results showed that compared with the wild-type strain, the biofilm formed by ΔpilZ strain significantly increased the biofilm thickness, the number of bacteria, while the contents of β-polysaccharides and proteins in EPS were decreased and the settlement and metamorphosis of M. coruscus larvae were inhibited (p<0.05). There was no significant difference in c-di-GMP level, α-polysaccharide and lipid contents (p>0.05). In conclusion, P. marina pilZ gene knockout can regulate the bacterial biofilm and the content of EPS including β-polysaccharides and proteins contents, then inhibit the settlement and metamorphosis of M. coruscus larvae.
- pilZ /
- biofilm /
- Mytilus coruscus /
- settlement and metamorphosis /
- extracellular polymeric substances

HTML全文

图 1 pilZ基因缺失验证（A）和缺失前后基因簇（B）

Fig. 1 The pilZ gene deletion verification (A) and gene cluster (B) before and after knockout

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图 2 不同初始细菌密度下野生菌与ΔpilZ菌生物被膜对幼虫附着变态的影响（A）及生物被膜细菌密度的分析（B）

图中不同字母表示在该初始细菌密度下，P. marina野生菌与ΔpilZ菌生物被膜的附着变态率或细菌密度组间有显著差异（p<0.05）

Fig. 2 Effect of wild-type and ΔpilZ biofilms formed with different initial bacterial density on larval settlement and metamorphosis (A) and the analysis of biofilm bacterial density (B)

Bars with different letters in the figure indicate that at the initial bacterial density, there’s a significant difference between the rate of larval settlement and metamorphosis or the bacterial density of P. marina wild-type and ΔpilZ biofilms (p<0.05)

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图 3 菌落形态

Fig. 3 Colony morphology of bacteria

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图 4 野生型菌株和ΔpilZ菌株运动性（A）、生物被膜（C、D）和膜厚（B）

Fig. 4 Motility (A), biofilm (C, D) and biofilm thickness (B) of wild-type and ΔpilZ strains

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图 5 野生型菌株和ΔpilZ菌株生物被膜共聚焦扫描图像（A）和统计结果（B）

Fig. 5 Confocal scanning images (A) and statistical results (B) of biofilm of wild-type and ΔpilZ strains

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图 6 野生菌和ΔpilZ菌的胞内c-di-GMP水平

Fig. 6 The c-di-GMP levels of wild-type and ΔpilZ strains

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表 1 本研究使用的细菌菌株和质粒

Tab. 1 The strains and plasmids used in this study

菌株/质粒	相关特性	来源
菌株 P. marina ECSMB14103 E. coli WM3064 ΔpilZ 质粒	野生型 DAP缺陷型 pilZ基因敲除菌株	文献[18] 文献[20] 本研究
pK18mobsacB-ery pK18mobsacB-ery-pilZ	具有Kan和Ery抗性基因位点敲除pilZ基因的重组质粒	文献[19] 本研究

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表 2 构建ΔpilZ菌株所使用的引物及其序列

Tab. 2 Primers used to construct ΔpilZ strain and its sequences

引物	序列（5'-3'）
pilZ-up-F	CGGGATCCAGGTGAACTTGACCGAATA
pilZ-up-R	CGGAATTCGGTTAATCCTTTTATTTATT
pilZ-down-F	CGGAATTCTAAAAAACAGGCCCAATTTT
pilZ-down-R	AACTGCAGGACAATGCCTGAAATAGAAA
pilZ-SF	CCCTGTGGGTGTAGGTAA
pilZ-SR	CGTCGCGTGTATGAATAA
pilZ-LF	CGACCGTCACGACTTATC
pilZ-LR	TGTTCGCTGACACTTTGC

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表 3 激光共聚焦显微镜荧光染料信息

Tab. 3 Laser confocal microscopy fluorescence dye information

荧光染料名称	结合物质	工作液浓度/ （μg·mL⁻¹）	波长范围/ nm
碘化丙啶（PI）	死细菌	5	560～700
刀豆蛋白A（ConA-TMR）	α-多糖	944.8	552～578
荧光增白剂（CFW M2R）	β-多糖	189.0	254～432
DilC₁₈（5）油（DiD`oil）	脂类	7.94	648～670
异硫氰酸荧光素异构体I（FITC）	蛋白质	46.6	495～519

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