白术叶片DNA提取方法的优化蒋小刚,王华,周武先,由金文,张美德∗(湖北省农业科学院中药材研究所农业农村部中药材生物学与栽培重点实验室,湖北恩施445000)摘要[目的]优化白术叶片DNA提取方法,提取高质量DNA,用于分子生物学研究。[方法]以白术叶片为材料,比较常规CTAB法、改良CTAB法、SDS法、试剂盒法提取DNA的效果,并对改良CTAB法中的裂解温度、裂解时间、核分离液解离次数进行优化,用琼脂糖凝胶电泳和微量核酸测定仪检测DNA的浓度和纯度。最后通过ISSR-PCR验证优化的改良CTAB法提取DNA的效果。[结果]4种方法中,改良CTAB法提取白术叶片DNA效果最好。针对叶片的改良CTAB法的优化条件为裂解温度65℃、裂解时间20min、解离次数2次;ISSR-PCR验证结果表明扩增条带清晰稳定,无拖尾。[结论]优化后的改良CTAB法提取白术叶片DNA质量高,可用于白术种质资源遗传多样性分析。关键词白术;DNA;提取方法;改良CTAB法中图分类号R284文献标识码A文章编号0517-6611(2023)11-0128-04doi:10.3969/j.issn.0517-6611.2023.11.032开放科学(资源服务)标识码(OSID):OptimizationofDNAExtractionMethodforAtractylodesmacrocephalaLeavesJIANGXiao-gang,WANGHua,ZHOUWu-xianetal(KeyLaboratoryofBiologyandCultivationofHerbMedicine,MinistryofAgricul-tureandRuralAffairs,InstituteofChineseHerbalMedicines,HubeiAcademyofAgriculturalSciences,Enshi,Hubei445000)Abstract[Objective]TooptimizetheDNAextractionmethodofAtractylodesmacrocephalaleavesandextracthigh-qualityDNAformolecularbiologyresearch.[Method]A.macrocephalaleaveswereusedasmaterial,theDNAextractioneffectsoffourmethods(conventionalCTAB,modifiedCTAB,SDSandKitmethod)werecompared,andthecrackingtemperature,crackingtimeandtimesofnuclearseparationliquidex-tractioninthemodifiedCTABmethodwereoptimized,theconcentrationandpurityoftheextractedDNAweredetectedbyagarosegelelectro-phoresisandspectrophotometer.Finally,theeffectsofDNAextractedbythemodifiedCTABmethodafteroptimizationwasverifiedbyISSRanalysis.[Result]Amongthefourextractionmethods,themodifiedCTABmethodhadthebestextractioneffectsofDNA.TheoptimalextractionconditionsoftheimprovedCTABmethodofleaveswereasfollows:crackingtemperaturewas65℃,crackingtimewastwentyminutes,dissocia-tiontimeswastwic...
