根癌农杆菌介导的稻瘟病菌近等菌系的构建与初步应用  

Agrobacterium tumefaciens-Mediated Construction and Preliminary Application of Near-isogenic Isolates of Magnaporthe oryzae

作　　者：何余勇[1] 周惠萍[1] 吴兴兴[1] 吴毅歆[1] 王志远[1] 毛自朝[2] 何月秋[1,2] 

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]云南农业大学农学与生物技术学院,云南昆明650201

出　　处：《云南农业大学学报（自然科学版）》2010年第5期603-610,共8页Journal of Yunnan Agricultural University:Natural Science

基　　金：国家行业计划项目(200803008);国家“863”计划项目(2002AA245041);农业部“948”项目(2006-G61)

摘　　要：利用根癌农杆菌C58C1介导转化1株致病力极弱的野生型稻瘟病菌菌株CY2,成功地获得3000多个转化子,混合接种于携带25个不同抗病单基因的水稻近等基因系上,获得4株单胞致病突变体;继代培养和PCR检测表明,这些突变体均为T-DNA插入CY2的后代,而非自发突变或外源菌株污染。将4株突变体与实验室已保存的20株致病突变体分别回接水稻近等基因系,推断其携带的致病基因类型,结果表明突变体分别针对13个抗性单基因的18个单基因系发生了突变,其中针对Pi-a,Pik-s,Pi-sh单基因系表现出较高的侵染频率。由于这些突变体均来自同一个菌株,仅致病性存在差异,属于近等菌系,利用其分析出102个水稻品种中的34个品种的抗病基因组成,其中,抗病基因Pik-s,Pi-a,Pi-sh,Pi-19(t)分布频率较高,分别达70.59%,61.76%,52.94%和44.12%,且带有这类基因的糯稻品种多于杂交水稻。Magnaporthe oryzae CY2 with weak pathogenicity to rice was transformed by Agrobacterium tumefaciens C58C1 mediation,and more than 3000 transformants were obtained,then 4 pathogenic mutants were screened out among them by inoculating onto the rice near-isogenic lines with 25 different resistance genes.The results of secondary cultures on the medium with hygromycin B and PCR amplification indicated the mutants were CY2＇s transformants by the insertion of T-DNA but not contaminated isolates.Then,4 pathogenic mutants plus other 20 pathogenic mutants in the laboratory were inoculated back onto the rice near-isogenic lines to investigate their different virulence genes,showing those mutants were pathogenic to rice lines with 13 different resistance genes,especially to the rice with Pi-a,Pik-s,Pi-sh genes.Due to the same origin and only pathogenicity difference,those mutants belong to near-isogenic isolates.They could detect rice resistance genes according to the gene-for-gene hypothesis.By the isolates,we investigated the resistance gene of 34 among 102 rice cultivars.The genes Pik-s,Pi-a,Pi-sh and Pi-19（t） were more common with 70.59%,61.76%,52.94% and 44.12% frequencies than others among 34 cultivars and more in glutinous than in hybrid rice.

关 键 词：稻瘟病菌 近等菌系 根癌农杆菌 T-DNA转化 抗病基因鉴定 

分 类 号：S435.111.41[农业科学—农业昆虫与害虫防治]