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期 刊 ：植物保护学报 2008年

关键词：稻瘟病菌；农杆菌介导遗传转化；T-DNA插入突变；

Keywords:Magnaporthe grisea, Agrobacterium-mediated transformation, T-DNA insertional mutagenesis,

摘 要 ：以农杆菌C58C1及携带潮霉素抗性的质粒pBIG2RHPH2为介导,以广泛不致病的野生型稻瘟病菌CY2为出发菌株,开展稻瘟病菌T-DNA插入转化条件的研究。农杆菌OD₆₀₀为0.1条件下,乙酰丁香酮(AS)200μmol/L,用IM培养基(pH5.2)共培养。结果表明,在潮霉素、头孢噻肟钠和壮观霉素为200μg/mL,2mm滤纸条筛选培养,转化效率最高,平均可获得329.0个转化子/1×10⁴个孢子。通过对转化子的继代稳定性和PCR检测,证明转化子均获得了T-DNA插入片段,且能稳定遗传。采用接种法,在不同遗传背景的44个抗稻瘟病的水稻近等基因系接种8000个转化子,获得20个致病突变体。

Abstract:T-DNA insertional transformation system of a universal non-pathogenic Magnaporthe grisea isolate CY2, was improved with Agrobacterium tumefaciens strain C58C1 carrying plasmid pBIG2RHPH2 harboring the hygromycin B gene. The result showed that total 329 transformants could be obtained per 1×10⁴ spores when C58C1 was 0.1 at OD₆₀₀, induction medium with pH5.2 containing acetosyringone at 200μmol/L, selection medium containing 200μg/mL of hygromycin B, cefotaxime sodium and spectinomycin each and 2mm wide filter paper stripe were used. The transformants were stable when they grew on CM medium without hygromycin B for 5 times and were verified with PCR amplification based on the primer pairs of the hygromycin resistance gene. By artificial inoculation on 44 near-isogenic lines with different backgrounds, 20 pathogenic mutants were isolated out of 8000 transformants.

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