荧光标记基因转化棉花黄萎病菌及标记菌系选育详细分析.doc

荧光标记基因转化棉花黄萎病菌及标记菌系选育 王晓楠袁齐宏袁张金凤袁光杨其袁唐灿明* 渊南京农业大学农学院/作物遗传与种质创新国家重点实验室袁江苏南京210095冤 摘要院为了研究黄萎病菌的侵染机制袁通过农杆菌介导的转化方法袁将绿色荧光蛋白基因导入落叶型黄 萎病菌VD07038袁将红色荧光蛋白基因导入非落叶型黄萎病菌Bp2中,分别获得了具有绿色尧红 色荧光信号的阳性转化子遥经过分子验证和连续继代培养袁证明了这些转化子具有遗传稳定的对潮霉素的抗 性遥通过对转化子的菌落形态尧生长速度和致病力进行检测袁发现大部分转化子与野生型基本一致袁少量转化 子发生变异袁其中转化子Bp2R-30不能产生微菌核袁致病力显著下降遥利用荧光显微镜观察了转化子 VD07038G-10在感病棉花品种苏棉22幼苗根部的侵染情况遥结果表明袁在接菌12h后VD07038G-10的孢子 可吸附在根表面曰接种7～9d后袁菌丝入侵到棉花根部的维管组织遥本研究获得的荧光蛋白标记的棉花黄萎 病菌VD07038G-10可用于实时观测黄萎病菌侵染棉花根系的过程袁并且可以定量鉴定不同棉花品种对该菌系 的抗性袁为棉花黄萎病菌抗性鉴定提供一种新方法遥 关键词院棉花曰大丽轮枝菌曰绿色荧光蛋白曰红色荧光蛋白曰转化 中图分类号院S435.621文献标志码院A 文章编号院1002-7807渊2014冤03-0221-07 WangXiaonan,QiHong,ZhangJinfeng,GuangYangqi,TangCanming* ( ,210095,) Inordertoanalysetheinfectionmechanismof,encodinggreenfluorescentproteinwastransferredin- todefoliatingisolateVD07038andencodingredfluorescentproteinwastransferredintonon-defoliating Bp2via-mediatedtransformation.Finally,positivetransformantstaggedbygreenfluores- centproteinorredfluorescentproteinwereobtained.ThehygromycinBgeneticstabilityoftransgenicprogenieswastestedby seriescultureandmolecularverification.Accordingtocolonymorphology,growthrateandpathogenicity,wefoundthattraitsof mostoftransformantsweresimilartothoseofwild-type,fewtransformantstendedtoformmutants.TansformantsBp2R-30did notproducemicrosclerotiaanditspathogenicitydecreasedsignificantly.UsingtheGFP-taggedVD07038G-10iso- late,colonizationofthefungusinrootsofthesusceptiblecottonSumian22wasinvestigated.Astheresultsshowedthat,12 hoursafterinoculationviatherootdippingmethods,rootssurfaceofSumian22werecoveredwithconidia,7-9daysafterinocu- lation,hyphaefromtheleadingedgeofthecolonyprogressedacropetallyupthexylemvesselsofinfectedroots. VD07038G-10taggedbyfluorescentproteincanbeusedforobservationitsinfectionprocessinrealtimeoncottonrootsandi- dentificationofresistanceofdifferentcottonvarietiestothisstrain.Thisstudyprovideanewmethodforresistanceevaluationof . cotton;Kleb.;greenfluorescentprotein;redfluorescentprotein;transformation 收稿日期院2013-11-22作者简介院王晓楠渊1988-冤袁女袁硕士