根癌农杆菌介导的水稻纹枯病菌转化系统的建立  

Establishment of Agrobacterium tumefaciens-Mediated Transformation System for Rhizoctonia solani AG-1ⅠA,the Causal Agent of Rice Sheath Blight

作　　者：杨迎青[1] 杨媚[1] 李明海[1] 李勇[1] 贺晓霞[1] 周而勋[1] 

机构地区：[1]华南农业大学植物病理学系,广东广州510642

出　　处：《中国水稻科学》2010年第6期617-622,共6页Chinese Journal of Rice Science

基　　金：国家公益性行业(农业)科研专项经费资助项目(ny-hyzx3-16;原nyzx07-049)

摘　　要：为建立水稻纹枯病菌的T-DNA插入诱变转化系统,以水稻纹枯病菌(Rhizoctonia solani AG-1ⅠA)强致病力菌株GD118为转化的初始菌株,从预诱导时间、共培养时间、共培养时乙酰丁香酮的浓度、共培养温度和共培养时固体诱导培养基(SIM)的pH值等5个方面对转化条件进行了优化,成功地建立了适合于水稻纹枯病菌根癌农杆菌介导转化(ATMT)的优化系统。这个优化系统的转化条件如下:以30μg/mL的潮霉素B作为转化子的筛选浓度,预诱导8h,共培养20h,共培养时固体诱导培养基上的乙酰丁香酮浓度为200μmol/L,共培养温度25℃,共培养时固体诱导培养基pH5.6～5.8。采用这个系统筛选到的转化子继代培养5代后,在含30μg/mL潮霉素B的PDA平板上仍表现明显的抗性。从得到的转化子中随机抽取10个,利用根据抗潮霉素hph基因设计的特异性引物进行PCR扩增,转化子均能扩增出500bp左右的预期条带;与此同时,以4个根癌农杆菌作为阳性对照,用根癌农杆菌Vir基因特异引物对转化子进行PCR扩增,以排除转化子受农杆菌污染所致的假阳性,结果表明:4个根癌农杆菌均能扩增出Vir基因条带(730bp),而10个转化子均未能扩增出相应条带。以上两个PCR扩增的实验结果清楚地表明,T-DNA已经插入到目标菌株GD118中。In order to construct the T-DNA insertional mutagenesis transformation system for rice sheath blight pathogen Rhizoctonia solani AG-1ⅠA,the virulent isolate GD118 of this pathogen was selected as the initial isolate for transformation.The conditions for the transformation of GD118 were optimized in 5aspects,i.e.pre-induction time,coculture time,acetosyringone concentration at coculture stage,coculture temperature and pH values of solid induction medium（SIM）at coculture phase.Finally,an Agrobacterium tumefaciens-mediated transformation（ATMT）system for R.solani AG-1ⅠA was established successfully.The optimal conditions for this ATMT system are as follows：the concentration of hygromycin B at 30μg/mL for transformant screening,8hof pre-induction time,20hof coculture time,200μmol/L of acetosyringone in SIM at coculture stage,coculture temperature at 25℃and pH 5.6to 5.8of SIM at coculture phase.The transformants still showed high resistance to hygromycin B after 5generations＇subcultures.Ten transformants were randomly picked out for PCR verification using the specific primers designed from the hphgene,and the results revealed that an expected band of 500bp was amplified from all of the 10transformants.Meantime,PCR amplification for these 10transformants was carried out using specific primers designed from the Vir gene of A.tumefaciens,with 4strains of A.tumefaciens as positive controls for eliminating the false-positive caused by A.tumefaciens＇s contamination,and the results showed that an expected band of 730bp could be amplified from the 4strains of A.tumefaciens,whereas no corresponding DNA band could be amplified from the 10transformants.The results of above two PCR amplifications clearly showed that T-DNA was indeed inserted into the targeted isolate GD118.

关 键 词：水稻纹枯病 立枯丝核菌 根癌农杆菌介导转化 T-DNA插入诱变 方法 

分 类 号：S435.111.4[农业科学—农业昆虫与害虫防治]