红鳍东方源哈维弧菌毒力基因检测及分型的研究.docVIP

红鳍东方源哈维弧菌毒力基因检测及分型的研究 　　摘要 为探明红鳍东方?源哈维弧菌所携带毒力基因的种类对致病性的影响，以从辽宁地区患病红鳍东方?中分离得到的24株哈维弧菌为材料，根据GenBank公布的基因序列，设计合成引物，采用聚合酶链式反应（Polymerase Chain Reaction，PCR）对7种毒力基因flaB、flaC、toxS、zot、tcpA、tdh和tlh进行检测。结果表明，24株哈维弧菌中均检测出鞭毛蛋白flaB基因和flaC基因，其余5种基因均未检测到。同时对24株哈维弧菌进行了BOX-PCR扩增，结果将其分为4种基因型，记为A1-A4型。 　　关键词 红鳍东方?；哈维弧菌；毒力基因；BOX-PCR 　　中图分类号 S965.225 文献标识码 A 文章编号 1007-5739（2018）02-0237-03 　　Virulence Genes Detection and BOX-PCR Identification of Vibrio harveyi Strains Isolated from Takifugu rubripes 　　WANG Li 1 ZHANG Ji-peng 1 LIU Mei-ru 1 YE Shi-gen 1 LI Rui-jun 1 LI Hua 1 LI Qiang 2 　　（1 Key Laboratory of Mari-cultureStock Enhancement in North China Sea，Ministry of Agriculture，Dalian Ocean University，Dalian Liaoning 116023； 2 College of Marine and Biology Engineering，Yancheng Institute of Technology） 　　Abstract In this paper，we detected virulence genetypes of Vibrio harvey isolated from Takifugu rubripes for further studying its pathogenicity.24 strains of V.harveyi isolated from infected Takifugu rubripes in Liaoning region were used for research.The PCR-primers were designed and synthesized according to the published sequence in the GenBank data.Polymerase Chain Reaction（PCR）was used to detect 7 virulence genes，including flaB，flaC，toxS，zot，tcpA，tdh and tlh.The results showed that both flaB and flaC gene were detected in all 24 strains of V.harveyi，and other 5 virulence genes were not detected.Meanwhile，the results of BOX-PCR indentation showed that 24 strains of V.harveyi were divided into 4 genotypes，respectively named A1-A4. 　　Key words Takifugu rubripes；Vibrio harvey；virulence gene；BOX-PCR 　　哈?S弧菌（Vibrio harveyi）属于革兰氏阴性菌，已成为水产养殖上的主要致病菌，被哈维弧菌感染的对象有对虾[1]、大黄鱼[2]、鲈鱼[3]、石斑鱼[4]、半滑舌鳎[5]、大菱鲆[6]、?|鱼[7]、鲍[8]、红鳍东方?[9]等水产动物，严重危害着养殖业的发展。随着分子生物学和免疫学技术的发展，哈维弧菌致病因子受到广泛的研究。研究表明，哈维弧菌内存在flaB、flaC、toxS、zot、tcpA、tdh和tlh基因，并且毒力基因的分布与分离菌株的来源有关[10-11]。同时，BOX-PCR指纹分析技术可以反映菌株间基因组的差异，广泛应用于生物多样性的研究[12]。因此，为了进一步探究哈维弧菌的致病机制，本文在原有工作的基础上，对辽宁地区患病红鳍东方?内分离鉴定的24株哈维弧菌进行flaB、flaC、toxS、zot、tcpA、tdh和tlh 7种毒