稻瘟病菌T-DNA插入方法优化及其突变体分析

Optimization of T-DNA Insertional Mutagenesis and Analysis of Mutants of Magnaporthe grisea

优化了农杆菌介导转化稻瘟病菌获得T DNA插入突变的条件 ,包括选择转化子的潮霉素B用量 ,抑制农杆菌的抗生素头孢噻肟钠和羧苄青霉素的配比 ,不同转化阶段培养基的选择等。转化 1× 10 6 个孢子平均可获得约5 0 0个左右的转化子 ,PCR和TAIL PCR检测表明约 85 %转化子中含T DNA插入。对 15 2 0个突变体进行形态变异观察 ,发现菌落颜色突变的有 15个 ;随机取 5 8个突变体进行比较 ,发现产孢量减少的 4个 ,孢子萌发率降低的 8个 ,附着胞形成率降低的 9个 ;还获得对水稻品种C10 1LAC(Pi 1)和 75 1 12 7(Pi 9)致病的突变体 。

The rice blast fungus Magnaporthe grisea causes one of the most destructive diseases of rice around the world. Significant progresses have been made recently in genomics studies of the fungus, opening new era of the functional genomics which requires to generate a large scale of gene knockout mutants. It has been demonstrated that T-DNA insertional mutagenesis is a powerful tool of functional genomics not only for plants but also for fungi. In this paper, we optimized the conditions for T-DNA insertional mutagenesis of M.grisea using Agrobacterium tumefaciens-mediated transformation (ATMT) approach. We employed the binary vector pBHt1 constructed by Dr. S. Kang's laboratory at the Pennsylvania State University, which carries the bacterial hygromycin B phosphotransferase gene (hph) under the control of the Aspergillus nidulans trpC promoter as a selectable marker to transform the conidia of M.grisea. We optimized the conditions for T-DNA insertional mutagenesis including the medium, dosage of hygromycin B, cefotaxime and carbenicillin to select the transformants and inhibit the growth of A.tumefaciens after co-culturing. The dosage to inhibit non-transformants could vary from 200-600μg/mL among different M.grisea isolates so that the optimal dosage of the antibiotics should be decided according to isolates. Rice polished agar medium was found the best selection medium which would facilitate the mutant sporulation and minimize the contamination chance. In average, about 500 transformants could be obtained when transforming 1*10 6 spores at the optimum condition, among which 85% had T-DNA insertion detected by polymerase chain reaction (PCR) and thermal asymmetric interlaced polymerase chain reaction (TAIL-PCR). Fifteen out of 1520 transformants showed mutation in colony morphology. Within 58 randomly selected mutants, it was found that there were 4 sporulation-decreased mutants, 8 less germination mutants and 9 appressorium defective mutants. Several virulent mutants to C101LAC(Pi-1)and 75-1-127(Pi-9)were also obtained which would facilitate cloning the corresponding avirulence genes.