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以甲基磺酸乙酯(ethylmethane sulfonate,EMS)为诱变剂,通过其对大豆疫霉菌Phytophthora sojae休止孢萌发的影响,确定化学诱变条件。通过收集单卵孢子,建立了包含640个单卵孢子系的突变体库,其中约有50%的诱变菌系在培养性状和菌落形态方面发生了明显变化,菌落形态多样,表现出较紧密或松散,近圆形或不规则;气生菌丝减少,生长速度较慢或快;在卵孢子产量方面,8.13%的菌系有增加,20.41%的菌系减少,27.82%的菌系极少或者没有卵孢子产生,43.64%的菌系卵孢子产量类似野生型。以质膜氢离子泵蛋白基因PsPMA1(plasma membrane H+-ATPase1)为对象,通过TILLING技术,从320个大豆疫霉菌突变体中获得9个突变体,进一步确认了EMS对大豆疫霉菌的诱变效果,并且估算EMS对大豆疫霉菌的诱变频率至多每115kb发生一个核苷酸变异。新构建的突变体库为开展大豆疫霉病菌的功能基因组研究奠定了遗传材料基础。
By using ethylmethane sulfonate (EMS) as a mutagen, the chemical mutagenesis conditions were determined by its effect on the spore germination of Phytophthora sojae. By collecting single oospores, a mutant library containing 640 single sporozoites was established, in which about 50% of mutagenic strains had obvious changes in culture and colony morphology. Colonies varied in morphology and showed more compact Or loose, nearly round or irregular; aerial mycelium decreased, the growth rate is slower or faster; in the production of oospores, 8.13% of the strains increased, 20.41% of the strains decreased, 27.82% Less or no oospores, 43.64% of the fungal oospores yield similar to the wild type. Nine mutants from 320 Phytophthora sojae strains were obtained by TILLING technique using plasma membrane H + -ATPase1 as a plasma membrane hydrogen plasma pump protein gene, further confirming the mutagenic effect of EMS on Phytophthora sojae , And estimated that the frequency of mutagenesis by EMS to Phytophthora sojae was at most one nucleotide variation per 115 kb. The newly constructed mutant library laid the foundation of genetic material for the functional genomics research of Phytophthora sojae.