基于ITS序列分析的外生菌根真菌的分子鉴定研究 生物技术专业VIP免费

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；鉴定题目：基于ITS序列分析的外生菌根真菌的分子鉴定研究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目录引言..............................................................11材料与方法........................................................31.1实验材料........................................................31.2实验设备及药品..................................................31.2.1试剂配方......................................................31.3实验方法........................................................31.3.1基因组DNA的提取.............................................31.3.2rDNAITS区段的PCR扩增......................................41.3.3PCR扩增产物的回收和纯化.....................................51.3.4DNA测序分析.................................................52结果与分析........................................................62.1PCR产物的检测.................................................62.2提纯效果的检测..................................................62.3rDNAITS序列的同源性分析.......................................72.4raxmlGUI系统发育树分析.........................................93结论与讨论.......................................................10参考文献...........................................................11致谢............................................................121引言贺兰山坐落于宁夏回族自治区的西北部，是银川平原的原始屏障，也是三北防护林建设的重点地段[1]。贺兰山植被类型比较复杂，既有标志山地所在水平地带属性的草原和荒漠，也有山地植被垂直系列中出现的针叶林和稀疏草原，还有各种灌丛、草甸和落叶阔叶林[2]。贺兰山是我国西北部温带草原与荒漠的分界线，同时也是连接蒙古高原、青藏高原及华...