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.
出处
《植物保护学报》
CAS
CSCD
北大核心
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)
