基因枪介导小麦条锈菌毒性突变的研究

Mutations induced by biolistics affecting virulence in Puccinia striiformis

为了得到带有明确遗传标记的毒性突变菌株,应用基因枪转化技术,以小麦条锈菌野生白化菌系为转化受体,以含有潮霉素抗性基因(Hyg)的质粒为插入载体,对基因枪介导小麦条锈菌插入突变的条件进行了研究。结果表明,当小麦条锈菌夏孢子萌动2 h,可裂膜承载压力为1 100 Psi时进行轰击转化,所得的小麦条锈菌夏孢子在含有潮霉素的培养基上萌发率最高,为36.09%。进一步将转化的小麦条锈菌夏孢子进行扩繁,在筛选品种上筛选,将筛选到的突变体在其上继代培养4代,获得了两个稳定的毒性突变菌株MM-2、MM-3。MM-2菌株对南大2419的毒性减弱,反应型由野生菌系的4型变为2型;MM-3菌株在南大2419的反应型由野生菌系的4型变为2、3型,毒性发生分化。采用中国鉴别寄主对突变体的毒性研究表明,各突变菌株的毒性均较原始菌系发生了明显改变。进一步用Hyg基因特异PCR在两个突变菌系中均扩增到目的片段,表明MM-2、MM-3的突变是由Hyg基因插入引起的。

In order to obtain the virulunce with genetic tag,insertional mutagenesis was made in Puccinia striiformis by using biolistics with the wild virulent albino strains of P. striiformis as transformation acceptors and the plasmids with gene conferring resistance to hygromycin B as vectors. Result showed germination rate of transformed urediospores on media containing hygromycin B reached the highest rate of 36.09 % when the transformation was hydrated 2 hours before bombardment of albino strain urediospores and under the delivery pressure of 1 100 Psi. The transformants were further propagated and stably selected on screen cultivars for four generations, through which we obtained two virulence mutants of strains. Virulence of MM2 isolate generated by non-virulence mutation on Mentana host was reduced, and transformed to reaction type 2 ,different from the original reaction type 4;MM3 isolate transformed to type 2 and 3 on Mentana host, from its original race Type 4,which was indicative of virulence differentiation. The virulence spectrum of mutants on China differential hosts indicated that significant virulence changes had occurred in each mutant in comparison with original races. Fragments of interest were also obtained by PCR amplification of two virulence mutants of strains,which showed MMZ and MM3 were mutated by Hyg gene.