根癌农杆菌介导柱花草炭疽菌遗传转化体系的优化

摘要

选取含有氯嘧磺隆抗性标记的ILV1基因和报告基因GFP二元载体的农杆菌AGL-1,采用单因子和双因子方法研究根癌农杆菌AGL-1介导柱花草炭疽菌CH008转化过程中各主要因素对转化率的影响,优化构建ATMT转化柱花草胶孢炭疽菌体系条件,使转化效率达到300 ～400个转化子/106个炭疽孢子,即根癌农杆菌AGL-1浓度OD600=0.8,炭疽菌分生孢子浓度为1x106个/mL,AS浓度为100 μmol/L,诱导时间为6h,共培养温度和时间分别为25 ℃和4d.经PCR检测都有GFP片段,并能够表达绿色荧光蛋白.这为进一步开展炭疽菌对柱花草的致病机理研究提供了依据,优化转化体系有利于后续突变体库的扩大、筛选突变体和致病功能基因的研究.%In this study,an Agrobacterium tumefaciens AGL-1 containing binary vector with ILV1 and GFP as report genes was used for transformation of Colletotrichum gloeosporioides.In order to gain higher transformation efficiency,the major facts affecting transformation efficiency was analyzed by a one-factor experiment and a twofactor experiment.The optimization of genetic transformation system of C.gloeosporioides was established,and we optimized the conditions of transformed system.The transformation efficiency was up to 300 to 400 transformants per 106 conidia.All the transformants tested had GFP gene comfirmed by PCR,and GFP signals were also detected.The study provided a basis for further study on the pathogenesis of C.gloeosporioides,optimized transformation system offered highly efficient means for generating an ATMT library of C.gloeosporioides.And it would help to analyze the function of pathogenicity gene of this pathogen.