西沙永乐群岛海域弧菌的分离鉴定

doi:10.14012/j.cnki.fjsc.2022.01.001

摘要/Abstract

摘要：

从西沙永乐群岛7个岛礁海域的海水中分离出47株弧菌,将47株菌株进行16S rDNA、HSP60和gyrB扩增,序列在Genebank中进行比对,构建系统发育树。结果显示利用16S rDNA和HSP60序列比对均无法鉴定弧菌到种。gyrB1和gyrB2引物均只能扩增出6株菌株的目标序列,gyrB3引物能扩增47株菌株的目标序列,且成功构建系统发育树,可鉴定弧菌到种。47株弧菌菌株分属于7个种,鸭公岛弧菌多样性最丰富,可分离到5种;石屿和羚羊礁筐仔沙洲弧菌多样性最低,均只分离到2种。本研究建立了可靠的海洋弧菌的分子鉴定方法,获得的菌株丰富了南海微生物菌种库,可为今后的研究与教学提供材料。

关键词: 弧菌, 鉴定, 16S rDNA, HSP60, gyrB

Abstract:

Forty-seven strains of Vibrio were isolated from sea area of 7 islands of Xisha Yongle Islands.The 16S rDNA,HSP60 and gyrB genes of the 47 strains were amplified,and their sequences were compared in GeneBank and then phylogenetic trees were constructed.The results showed that neither 16S rDNA nor HSP60 could be used to identify the species of Vibrio.Both gyrB1 and gyrB2 primers could only amplify the target sequence of 6 strains.gyrB3 primers could amplify the target sequence of all the 47 strains,and the phylogenetic trees were successfully constructed.Species could be identified using gyrB3 primers.The 47 strains of Vibrio were divided into 7 species.Five strains of Vibrio was isolated in Yagong Island which had the highest diversity.Two species were isolated in both Shiyu and Antelope reef which had the lowest diversity.In this study,a reliable molecular identification method for marine Vibrio was established.The obtained strains enriched the microbial library of the South China Sea and provided materials for future research and teaching.

Key words: Vibrio, identification, 16S rDNA, HSP60, gyrB

中图分类号:

张秀霞, 张泽龙, 李军涛, 鲁耀鹏, 郑佩华, 冼健安. 西沙永乐群岛海域弧菌的分离鉴定[J]. 渔业研究, 2022, 44(1): 1-8.

ZHANG Xiuxia, ZHANG Zelong, LI Juntao, LU Yaopeng, ZHENG Peihua, XIAN Jian’an. Isolation and identification of marine Vibrio from Xisha Yongle Islands,China[J]. Journal of Fisheries Research, 2022, 44(1): 1-8.

图/表 8

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目标基因 Target genes	引物 Primers	引物序列(5’-3’) Sequences(5’-3’)	产物大小/bp Amplification size
16S rDNA	16S-F(通用引物)	AGAGTTTGATCCTGGCTCAG	1 400~1 500
16S rDNA	16S-R(通用引物)	GGTTACCTTGTTACGACTT	1 400~1 500
HSP60	HSP60-F^[4]	ACAACAGCAACGGTACTAGC	500~550
HSP60	HSP60-R^[4]	CAACTTTCACGATGCCAC	500~550
gyrB	gyrB1-F^[17]	TCCGGCGGTCTGCACGGCGT	1 200~1 300
	gyrB1-R^[17]	TTGTCCGGGTTGTACTCGTC
	gyrB2-F^[5]	AAGTCATCATGACCGTTCTGCAYGCNGGNGGNAARTTYGA
	gyrB2-R^[5]	AGCAGGGTACGGATGTGGCGAGCCRTCNACRTCNGCRTCNGTCAT
	gyrB3-F^[5]	GAAGTTATCATGACGGTACTTC
	gyrB3-R^[5]	CCTTTACGACGAGTCATTTC

目标基因 Target genes	引物 Primers	引物序列(5’-3’) Sequences(5’-3’)	产物大小/bp Amplification size
16S rDNA	16S-F(通用引物)	AGAGTTTGATCCTGGCTCAG	1 400~1 500
16S rDNA	16S-R(通用引物)	GGTTACCTTGTTACGACTT	1 400~1 500
HSP60	HSP60-F^[4]	ACAACAGCAACGGTACTAGC	500~550
HSP60	HSP60-R^[4]	CAACTTTCACGATGCCAC	500~550
gyrB	gyrB1-F^[17]	TCCGGCGGTCTGCACGGCGT	1 200~1 300
	gyrB1-R^[17]	TTGTCCGGGTTGTACTCGTC
	gyrB2-F^[5]	AAGTCATCATGACCGTTCTGCAYGCNGGNGGNAARTTYGA
	gyrB2-R^[5]	AGCAGGGTACGGATGTGGCGAGCCRTCNACRTCNGCRTCNGTCAT
	gyrB3-F^[5]	GAAGTTATCATGACGGTACTTC
	gyrB3-R^[5]	CCTTTACGACGAGTCATTTC

菌落形态 Colony morphology	图示 Figure	代表菌株 Representative strains
扁平圆形,菌落干燥,表面光滑,边缘平整 The colonnies were flat,round and dry,and had smooth surface and flat edge		YG1-4(Y)、YG1-5(Y)、GQ2-2(Y)、 GQ2-3(Y)
扁平圆形,菌落湿润,表面光滑,边缘平整 The colonnies were flat,round and moist,and had smooth surface and flat edge		LY2-4(Y)、QF2-1(Y)、QF2-2(Y)、 JQ2-1(G)、GQ1-2(Y)
不规则圆形,菌落湿润,表面光滑,边缘平整,菌落中间有数个小突起 The colonnies were irregular circle and moist,and had smooth surface and flat edge.There were several small protuberances in the middle of the colonnies		YY1-7(Y)、JQ2-1(G)、QF2-4(Y)
形状不规则,表面突起,菌落湿润,扩散状,边缘皱褶 The colonnies were irregular shape,moist and diffused,and had protuberance surface and wrinkle edge		GQ1-1(G)、GQ2-4(G)、SY2-2(G)、 SY2-6(G)、QF1-1(G)、QF1-2(G)、 QF2-1(G)
形状不规则,菌落扁平,稍微扩散,湿润 The colonnies were irregular shape,moist and slightly diffused,and had smooth surface		LY2-3(G)、YG2-4(G)、YG2-5(G)、 QF2-5(G)、GQ1-5(G)
圆形,菌落湿润,中间凸起,凸起为墨绿色,边缘为青白色 The colonnies were round and moist.The colonies bulge in the middle.Middle of colonies were dark green while edge of colonies were light green		LY2-1(G)、LY2-2(G)

菌落形态 Colony morphology	图示 Figure	代表菌株 Representative strains
扁平圆形,菌落干燥,表面光滑,边缘平整 The colonnies were flat,round and dry,and had smooth surface and flat edge		YG1-4(Y)、YG1-5(Y)、GQ2-2(Y)、 GQ2-3(Y)
扁平圆形,菌落湿润,表面光滑,边缘平整 The colonnies were flat,round and moist,and had smooth surface and flat edge		LY2-4(Y)、QF2-1(Y)、QF2-2(Y)、 JQ2-1(G)、GQ1-2(Y)
不规则圆形,菌落湿润,表面光滑,边缘平整,菌落中间有数个小突起 The colonnies were irregular circle and moist,and had smooth surface and flat edge.There were several small protuberances in the middle of the colonnies		YY1-7(Y)、JQ2-1(G)、QF2-4(Y)
形状不规则,表面突起,菌落湿润,扩散状,边缘皱褶 The colonnies were irregular shape,moist and diffused,and had protuberance surface and wrinkle edge		GQ1-1(G)、GQ2-4(G)、SY2-2(G)、 SY2-6(G)、QF1-1(G)、QF1-2(G)、 QF2-1(G)
形状不规则,菌落扁平,稍微扩散,湿润 The colonnies were irregular shape,moist and slightly diffused,and had smooth surface		LY2-3(G)、YG2-4(G)、YG2-5(G)、 QF2-5(G)、GQ1-5(G)
圆形,菌落湿润,中间凸起,凸起为墨绿色,边缘为青白色 The colonnies were round and moist.The colonies bulge in the middle.Middle of colonies were dark green while edge of colonies were light green		LY2-1(G)、LY2-2(G)

地点 Locale	菌株数量 Number of strains	种 Species	种的数量 Number of species
晋卿岛 Jinqing Island	10	Vibrio alginolyticus、Vibrio harveyi、Vibrio mediterranei	3
石屿 Shiyu Island	5	Vibrio alginolyticus、Vibrio coralliilyticus	2
银屿 Yinyu Island	5	Vibrio alginolyticus、Vibrio owensii、Vibrio fortis	3
全富岛 Quanfu Island	6	Vibrio alginolyticus、Vibrio owensii、Vibrio harveyi	3
鸭公岛 Yagong Island	9	Vibrio alginolyticus、Vibrio harveyi、Vibrio mediterranei、Vibrio owensii、Vibrio variabilis	5
甘泉岛 Ganquan Island	7	Vibrio alginolyticus、Vibrio coralliilyticus、Vibrio mediterranei、Vibrio variabilis	4
羚羊礁 Antelope reef	5	Vibrio alginolyticus、Vibrio harveyi	2