病原哈维氏弧菌(Vibrio harveyi)TS-628菌株基因组分析

doi:10.14012/j.cnki.fjsc.2023.05.001

摘要/Abstract

摘要：

哈维氏弧菌是海洋鱼类和无脊椎动物的重要条件致病菌,经常造成海水养殖动物严重病害,给水产养殖业造成巨大的经济损失,而不同来源的哈维氏弧菌的毒力存在很大差异。本研究采用第二代 Illumina Hiseq 平台与第三代 PacBio 平台相结合的测序技术,对分离自福建海域患病青石斑鱼的哈维氏弧菌TS-628菌株进行基因组测序和分析,挖掘该菌的基因组信息,从基因组层面了解该病原菌的毒力相关基因,以期为哈维氏弧菌后续致病机制的研究以及哈维氏弧菌病防治提供有力数据支撑。结果表明,哈维氏弧菌TS-628菌株基因组含2条染色体、2个质粒,其中1个质粒为新发现质粒;基因组大小为6 151 198 bp,基因组GC含量为44.68%,共编码5 658个基因,共预测到毒力基因747个,毒力基因主要与细菌的黏附、摄铁系统及分泌系统有关;此外,还发现耐药基因294个,反映该菌株不仅具有编码多种毒力因子的潜力,同时具有抗多种药物的可能。

关键词: 哈维氏弧菌, 基因组, 毒力基因, 基因组测序

Abstract:

V.harveyi is an important opportunistic pathogen of marine fish and invertebrates,which often causes serious diseases of mariculture animals and brings huge economic losses to the aquaculture industry.However,the virulence of V.harveyi isolated from different environments varies greatly.In the present study,the sequencing technology combining the second generation Illumina Hiseq platform and the third generation PacBio platform was used to sequence and analyze the genome of V.harveyi strain TS-628 isolated from the diseased Epinephelus awoara.The genome information of this strain was exploited to understand the virulence related genes of the pathogen,in order to provide data support for the subsequent research on the pathogenesis of V.harveyi and control of V.harveyi disease.The results revealed that the genome of V.harveyi strain TS-628 had two chromosomes and two plasmids.One of the plasmids was a newly discovered plasmid.The genome size of this strain was 6 151 198 bp,the GC content of the genome was 44.68%,the number of predicted genes were 5 658,the number of predicted virulence genes were 747.The 747 virulence genes were mainly related to bacterial adhesion,iron uptake and secretion systems.In addition,294 drug-resistant genes were found,reflecting that the V.harveyi strain TS-628 not only had the potential to encode multiple virulence factors,but also had the potential to resist multiple antibacterial agents.

Key words: Vibrio harveyi, genome, virulence gene, genome sequencing

中图分类号:

S917.1

李辰睿, 徐荣亮, 张小旭, 陈东萍, 邱于洋, 陈伟琴, 覃映雪. 病原哈维氏弧菌(Vibrio harveyi)TS-628菌株基因组分析[J]. 渔业研究, 2023, 45(5): 415-426.

LI Chenrui, XU Rongliang, ZHANG Xiaoxu, CHEN Dongping, QIU Yuyang, CHEN Weiqin, QIN Yingxue. Genome analysis of pathogenic Vibrio harveyi strain TS-628[J]. Journal of Fisheries Research, 2023, 45(5): 415-426.

图/表 12

图1 菌株TS-628 的 GC含量与测序深度分布图注:散点图的横坐标为GC含量,纵坐标为reads覆盖深度;右、上两侧直方图,分别为GC含量、测序深度的滑窗频数分布。

Fig.1 GC% and sequencing depth profiles of strains TS-628 Notes:The abscissa of the scatter chart is the GC content,and the ordinate is the read coverage depth;the histogram at right and up sides shows the frequency distribution of sliding window of GC content and sequencing depth,respectively.

表1 TS-628菌株基因组组装结果

Tab.1 The genome assembly results of strains TS-628

样品名称 Sample name	染色体数量 Chromosome no.	质粒数量 Plasmid no.	基因长度/bp Genome size	GC含量/% GC content
哈维氏弧菌 V.harveyi	2	2	6 151 198	44.68

表2 TS-628菌株基因组组装详情

Tab.2 The genome assembly details of strains TS-628

程序集 Assembly ID	序列号 Sequence ID	样品名称 Sample name	长度/bp Length	GC含量/% GC content	组成信息 Bases statistics	序列类型 Sequence type
染色体1 Chromosome 1	Scaffold 1	V.harveyi	3 574 667	44.95	A:980761;T:986964; G:80357;C:803371	Chromosome
染色体2 Chromosome 2	Scaffold 2	V.harveyi	2 292 642	44.76	A:633107;T:633402; G:514578;C:511555	Chromosome
质粒A Plasmid A	Scaffold 3	V.harveyi	217 554	39.41	A:61456;T:70356; G:43418;C:42324	Known-Plasmid
质粒B Plasmid B	Scaffold 4	V.harveyi	66 335	44.56	A:17821;T:18954; G:15310;C:14250	New-Plasmid

图2 基因组 CGView图注:图2A为染色体1;图2B为染色体2;图2C为质粒A;图2D为质粒B。从内圈到外圈,第1圈代表基因组全长;第2圈表示GC skew,其中绿色表示G-C>0,紫色则相反,绿色和紫色的交接处分别表示复制起始点和终止点;第3圈黑色表示GC含量,峰值向外突出的部分表示该区域GC含量高于全基因组平均GC含量,向内则相反;第4圈和第5圈分别表示正、负链上的基因,其中包括tRNA、rRNA、CDS和其他基因。

Fig.2 The CGView map of genome Notes:Figture 2A is chromosome 1;Figture 2B is chromosome 2;Figture 2C is plasmid A;Figture 2D is plasmid B.From the inner circle to the outer circle,the first circle represents the total length of the genome;the second circle represents the GC skeleton,where green represents G-C>0,while purple represents the opposite,the intersection of green and purple represents the start point and end point of replication,respectively;the black in the third circle indicates the GC content,and the protruding part of the peak indicates that the GC content in this region is higher than the average GC content of the whole genome,while the opposite is true inward;the fourth and fifth circles respectively represent genes on the positive and negative chains,including tRNA,rRNA,CDS and other genes.

图3 编码基因长度分布图

Fig. 3 The length distribution map of coding gene

表3 毒力基因预测分类表

Tab.3 The prediction and classification of virulence genes

样品名称 Sample name	类别1 Level 1	类别2 Level 2	基因数量 Gene no.	类别1 Level 1	类别2 Level 2	基因数量 Gene no.
	防御性毒力因子	应激蛋白	13	攻击性毒力因子	入侵类	13
哈维氏弧菌 V.harveyi	非特异性毒力因子	铁吸收系统	130	防御性毒力因子	相位变异	9
	防御性毒力因子	抗吞噬类	36	防御性毒力因子	抗血清	11
	毒力调控相关基因	调控相关	35	非特异性毒力因子	镁吸收系统	4
	攻击性毒力因子	分泌系统	107	防御性毒力因子	补体蛋白酶	2
	攻击性毒力因子	附着类	132	攻击性毒力因子	毒素	30

图4 毒力基因预测分类图

Fig. 4 The prediction and classification of virulence genes

表4 分泌系统分类统计表

Tab.4 The classification and statistics of secretory system

样品名称 Sample name	类型1 Type Ⅰ	类型2 Type Ⅱ	类型3 Type Ⅲ	类型4 Type Ⅳ	类型5 Type Ⅴ	类型6 Type Ⅵ	Sec-SRP型 Sec-SRP	Tat型 Tat	基因总数 Total gene no.
V.harveyi	1	12	17	1	0	7	13	3	54

图5 分泌系统通路图注:分泌系统通路图对每个样本中所有分泌系统的图示化展示。其中红色框标记的是在该样本中预测到的分泌系统相关基因。

Fig.5 The secretory pathway Notes:The secretory system pathway diagram is a graphical representation of all secretory systems in each sample.The red box marks the genes related to the secretion system predicted in the sample.

表5 双组分调控分类统计表

Tab.5 The statistics of two-component regulation classification

样品名称 Sample name	调控基因数量 Gene no.of regulator	传感基因数量 Gene no.of sensor	混合基因数量 Gene no.of hybrid
V.harveyi	149	63	30

图6 耐药基因预测分类统计图

Fig.6 The prediction and classification of drug resistance genes

表6 3株哈维氏弧菌主要毒力因子比较分析表

Tab.6 Comparative analysis table of the main virulence factors of 3 strains of V.harveyi

菌株 Strains	菌株来源 Source	基因长度/bp Genome length	GC 含量/% GC content	染色体数量 Chromosome no.	质粒数量 Plasmid no.	主要毒力基因种类 Major virulence gene species
TS-628(PRJNA874896)	青石斑鱼	6 151 198	44.68	2	2	附着类、铁摄取、分泌类
QT-520(PRJNA340970)	卵形鲳鲹	6 070 846	45.05	2	3	附着类、抗吞噬类、毒素
345(PRJNA418027)	龙胆石斑	6 185 822	44.76	2	2	附着类、铁摄取、运动性、分泌类

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