农杆菌介导的拟康氏木霉遗传转化及T-DNA插入突变体的筛选

Agrobacterium tumefaciens-mediated transformation of Trichoderma pseudokoningii SMF2 and isolation of the T-DNA insertion mutant

建立并优化了农杆菌介导转化拟康氏木霉（Trichoderma pseudokortingii SMF2）获得T-DNA插入突变体的体系，在木霉孢子浓度10^6个／mL、农杆菌A600=0．15～0．20、乙酰丁香酮浓度为250μmol／mL的条件下共培养36h转化率最高，转化子可达60-120个／10^5个孢子。共获得转化子1000多个，连续转接5代能够稳定遗传，部分转化子表现为生长和形态异常，PCR验证潮霉素B抗性基因已整合进转化子基因组DNA中。该转化体系的建立为开展其生防机理研究和探索菌株改良奠定了基础。

By using Agrobacterium turnefaciens-mediated transformation, we successfully transformed Trichoderma pseudokoningii SMF2 and obtained T-DNA insertion mutants. Under the optimal conditions （106spores/mL, A. tumefaciens A600=0.15-0. 20, 250 μmol/mL acetosyringone and 36 h co-cultivation）, the transformation efficiency reached 60-120 transformants per 106 spores. More than 1 000 transformants were obtained. Most of them were quite stable after five rounds of successive cultures, and some mutants were abnormal in morphology or spore formation. PCR amplification showed that the T-DNA had been integrated into the genome, and was stable through mitotic cell division.