一种农杆菌介导稻瘟病菌的遗传转化  

A genetic of transformation of Magnaporthe grisea by Agrobacterium tumefaciens

作　　者：吴毅歆[1,2] 范成明[1] 周惠萍[1] 久保康之 何月秋[1] 

机构地区：[1]云南农业大学农业生物多样性与病虫害控制教育部重点实验室,云南昆明650201 [2]云南省农业科学院经济作物研究所,云南昆明650205 [3]日本京都府立大学农学部植物病理实验室,日本京都606-8522

出　　处：《植物保护学报》2008年第5期421-426,共6页Journal of Plant Protection

基　　金：农业部“948”项目(2006-G61);国家科技部“863”计划(2002AA245041)

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)200μmol/L,用IM培养基(pH5.2)共培养。结果表明,在潮霉素、头孢噻肟钠和壮观霉素为200μg/mL,2mm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8000个转化子,获得20个致病突变体。T-DNA insertional transformation system of a universal non-pathogenic Magnaporthe grisea iso- late CY2, was improved with Agrobacterium tumefaciens strain C58C1 carrying plasmid pBIG2RHPH2 harboring the hygromyein B gene. The result showed that total 329 transformants could be obtained per 1×10^4 spores when C58C1 was 0.1 at OD600, induction medium with pH 5.2 containing acetosyringone at 200μmol/L, selection medium containing 200μg/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 8000 transformants.

关 键 词：稻瘟病菌 农杆菌介导遗传转化 T-DNA插入突变 基因系接种 

分 类 号：S435.111.41[农业科学—农业昆虫与害虫防治]