根癌农杆菌介导的轮枝镰孢菌遗传转化及T-DNA插入突变体的筛选

Agrobacterium tumefaciens-mediated transformation of Fusarium verticillioides and T-DNA insertional mutagenesis of the fungus

建立并优化了农杆菌介导转化轮枝镰孢菌Fusarium verticillioides获得T-DNA插入突变体的体系,在镰孢菌孢子浓度106个/mL、农杆菌OD600=0.15-0.20、乙酰丁香酮浓度为200μmol/mL的条件下共培养36h转化率最高,可达60-120个/106个孢子。共获得转化子1000多个,连续转接5代能够稳定遗传。PCR验证潮霉素B抗性基因已整合进转化子基因组DNA中,部分转化子表现为生长和形态异常。该转化体系的建立为研究该菌的致病机制和功能基因分析奠定了基础。

By using Agrobacterium tumefaciens-mediated transformation, we successfully transformed Fusarium verticillioides and obtained T-DNA insertion mutants. Under the optimal condition of 10^6spores/mL, A.tumefaciens OD600=0.15-0.20 200μg/mL acetosyringone and 36h co-cultivation, the transformation efficiency reached 60-120 transformants per 10^6spores. More than 1000 transformants were obtained. Most of them were quite stable after five rounds of successive cultures. PCR amplification showed that the T-DNA was integrated into the genome, and was stable through mitotic cell division. The transformation system is the basis for study of pathogenicity mechanism and functional gene of the fungi.