橡胶树胶胞炭疽菌T-900突变体生物学研究及其假定基因LV1敲除载体构建

摘要

从构建的橡胶树胶孢炭疽菌RC178(Colletotrichum gloeosporioides)突变体库中筛选获得一株致病力明显减弱突变体T-900,与野生菌株相比其菌落生长速率、产孢能力,孢子萌发率及附着孢形成率均明显降低.通过对突变菌株T-900 T-DNA侧翼序列克隆、比对和基因预测结果表明外源片段的插入破坏了一个预测基因的功能,该基因和组蛋白H3同源性为99％,暂命名为LV1.通过同源臂克隆构建了该基因敲除载体900-1A-900-1B-pCT74,为进一步研究该基因在病原菌致病过程中的作用机理提供了基础.%A mutant strain T-900 with weakened virulence to rubber trees was obtained from T-DNA insertion mutation library,and the conidia sporulation rate,spore germination and appressorium formation rate of T-900 were significantly lower than the wild-type stain RC178.The T-DNA flanking sequence of T-900 was obtained by TAIL-PCR and compared with the whole genome of C.gloeosporioides by sequence alignment.The results showed that the sequence in which the T-DNA was inserted contains a gene that was designated as LV1,The gene and histone H3 homology were 99％.In order to study the function of the pathogenicity gene LV1,The gene knockout vector 900-1A-900-1B-pCT74 was successfully constructed by homologous cloning and restriction endonuclease digestion.This method was successfully used to construct a high eflicient gene knockout system of LV1,which provides a technical platform for the functional verification of the pathogenic genes of Colletotrichum gloeosporioides.