论文部分内容阅读
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1×106个/mL,AS浓度为100μmol/L,诱导时间为6 h,共培养温度和时间分别为25℃和4 d。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。
Agrobacterium AGL-1 containing ILV1 and GFP binary vectors with chlorimuron-ethyl resistance markers was selected to study Agrobacterium tumefaciens AGL-1 mediated transformation of Colletotrichum gloeosporioides CH008 by single factor and two factor methods In order to optimize the conditions for the transformation of Colletotrichum gloeosporioides by ATMT, the transformation efficiency reached 300-400 transformants / 106 anthracnose spores, that is, the concentration of Agrobacterium tumefaciens AGL-1 OD600 = 0.8, the conidia concentration of anthracnose was 1 × 106 / mL, the concentration of AS was 100μmol / L and the induction time was 6 h. The co-culture temperature and time were 25 ℃ and 4 d respectively. GFP fragments are detected by PCR and can express green fluorescent protein. This provided the basis for further research on the pathogenic mechanism of Colletotrichum on Stylosanthes, optimizing transformation system is conducive to the expansion of the subsequent mutant library, screening of mutants and pathogenicity genes.