哈维氏弧菌ToxR部分基因序列克隆与分析【毕业论文】

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1、本科毕业设计（20__届）生物工程哈维氏弧菌ToxR部分基因序列克隆与分析摘要哈维氏弧菌是水产病害中主要的致病菌之一。根据已发表的哈维氏弧菌toxR基因片段，设计和合成一对上下游简并引物，应用聚合酶链式反应（PCR）方法，自哈维氏弧菌致病菌株基因组中扩增获得一段大小约384bp的ToxR基因片段，将获得的扩增片段进行鉴定和测序，并将测序结果与GenBank中已发表的哈维氏弧菌标准菌株的ToxR基因进行比对，同源性最高，达到了99%。与基因库中其他弧菌的相应序列对比显示，该序列与灿烂弧菌的ToxR基因有73%的同源性

2、，与溶藻弧菌的ToxR基因有78%的同源性，与溶珊瑚弧菌的ToxR基因有66%的同源性，与坎氏弧菌的ToxR基因有73%的同源性，与副溶血弧菌的ToxR基因有77%的同源性。该基因编码一个由116个氨基酸组成的蛋白质。用DNASTAR软件建立系统进化树并对其进行分析，证明这株菌为哈维氏弧菌，并且表明不同致病性弧菌的ToxR基因序列存在较大分歧，哈维氏弧菌与进化相近的坎氏弧菌和灿烂弧菌之间能比较容易被鉴别和区分开。ToxR基因序列的克隆与分析，为哈维氏弧菌的菌种鉴定提供了新的分子靶标，有助于其结构和功能的研究及通过突

3、变分析鉴定出新的受ToxR基因调控的致病基因。关键词：哈维氏弧菌；ToxR基因；基因克隆；序列分析ABSTRACTVibrioharveyiisoneofthemainpathogenicbacteriaofaquaticdisease.BasedonvibrioharveyiToxRgenesequenceswhichwaspublished,designingandsynthesizingapairofupstreamanddownstreamprimer.Wecangetawhileaboutthesizeo

4、f384bpToxRgeneticfragmentsbyaugmentingpathogenicvibrioharveyistraingenomeusingpolymerasechainreaction(PCR)method.Thanidentificationandsequencingtheamplificationfragmentswhichwegot,andcomprisingtheresultsofsequencingtothegeneToxRofthestandardvibrioharveyiwhichh

5、adpublishedinGenBand,have99%ofthehomology.SequenceanalysisandalignmentoftheToxRnucleotidesequencefromvibrioharveyiwiththosefromotherVibriospeciesrevealedthatthecompleteToxRgeneofvibrioharveyishares73%sequencesimilaritywiththeToxRgeneofVibriosp,78%withVibrioalg

6、inolyticus,66%withVibriocoralliilyticus,73%withVibriocampbellii,and77%withVibrioparahaemolyticus.Itencodeda116aaproteinasexpected.MakingaevolutionarytreebyDNASTARsoftwareandanalyzing,wecanprovingitisthevibrioharveyi,andrevealedawidedivergenceinToxRamongseveral

7、pathogenicvibrios,andvibrioharveyicouldbeeasilydistinguishedfromitsclosespeciesvibriosp.CloningandanalyzingoftheToxRsequence,whichmaybeagoodtargetforspeciesidentificationofvibrioharveyi,wouldpromotestructurefunctionstudiesandmutationanalysissoastoidentifyToxR-

8、regulatedgenes.Keyword:Vibrioharveyi;ToxR;geneclone;sequenceanalysis目录1前言12材料与方法22.1实验菌株22.2仪器22.3主要试剂22.4培养基22.5哈维氏弧菌总DNA的提取22.6ToxR基因PCR扩增32.7PCR产物的测序32.8扩增片段序列分析和ToxR基因系统发育学分析33