基于ITS序列分析的外生菌根真菌的分子鉴定研究.doc

包头师范学院本科毕业论文题目：基于ITS序列分析的外生菌根真菌的分子鉴定研究学生姓名：梁佳楠学院：生物科学与技术专业：生物技术班级：2014级生物技术指导教师：赵艳玲副教授二〇一八年五月摘要本实验是利用采自内蒙古贺兰山的外生菌根真菌子实体作为研究对象，通过常规CTAB法对该子实体进行基因组DNA提取，并利用真菌特有的引物ITS1F和ITS4对特异的DNA片段进行PCR扩增，将扩增产物在浓度为1%的琼脂糖凝胶中电泳进行检测，并在紫外透射反射分析仪下观察检测的结果，对于较好的扩增产物利用回收试剂盒进行纯化，后将纯化产物送由上海生工进行DNA测序，将测序所得到的序列输入GeneBank数据库中的局部相似性查询（BasicLocalAlignmentSearchTool,BLAST)程序进行比对，列出数据库中与所测得到的序列同源性较高的序列信息并进行分类鉴定，然后运用软件raxmlGUI进行系统发育树构建。最终的研究结果显示该菌种属于报道的choiromyceshelanshanensis。关键词：外生菌根真菌；ITS序列；分子生物学；鉴定AbstractInthisexperiment,anectomycorrhizalfungicollectedfromHelanMountain,InnerMongolia,wasusedastheresearchobject.TheextractionofgenomicDNAofthefruitingbodyofectomycorrhizalfungiwascarriedoutbytheconventionalCTABmethod.ThespecificprimersITS1FandITS4wereusedtoamplifythespecificDNAsequencesbyPCRamplification.Theamplifiedproductsweretestedbyelectrophoresiswitha1%agarosemolecularbiologygradegel,andtheresultsofthegelwereobservedundertheultraviolettransmissionreflectanceanalyzer.Thebetteramplificationproductswerepurifiedbyusingarecoverykit.Then,thepurifiedproductsweresenttoShanghaiShenggongforDNAsequencing.ThesequenceobtainedbysequencingwasinputintotheBasicLocalAlignmentSearchTool(BLAST)programintheGeneBankdatabaseforcomparsion.Inthedatabase,findoutthesequenceswithhigherhomologywiththesequencedsequencesandperformtheclassificationandidentification.ThephylogenetictreewasconstructedbyusingthesoftwareraxmlGUI.ThefinalresultsshowedthatthestrainbelongstothereportedChoiromyceshelanshanensisinHelanMountain,InnerMongolia.Keywords：ectomycorrhizalfungi;ITSsequence;molecularbiology;identification目录TOC\o"1-3"\h\z\uHYPERLINK\l"_Toc325043830"引言1HYPERLINK\l"_Toc325043831"1材料与方法3HYPERLINK\l"_Toc32504383