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本研究通过对菌核型强致病力菌株Vd080产生的800个T-DNA插入突变体进行致病力测定,筛选得到31株致病力极显著降低的突变体,并对其进行了生物学性状分析。分子验证结果表明,这些低致病力突变体均为阳性,且有25株的T-DNA为单拷贝插入。与野生型菌株Vd080相比,这25株低致病力突变体菌落形态变异丰富,除了8株与野生型菌株形态一致的菌核型突变体外,还有3株黄色菌丝型,2株白色菌丝型和12株中间型突变体。此外,多数突变体的生长速率、产孢量和粗毒素产量都发生了显著变化,且各生理指标之间并无明显相关性。借助TAIL-PCR技术和Vd Ls.17的基因组数据库,进一步获得了这25株单拷贝插入低致病力突变体T-DNA的侧翼序列,并从Vd080中成功克隆得到了影响黄萎病菌致病力的相关基因。
In this study, pathogenicity of 800 T-DNA insertion mutants produced by the strong virulence strain Vd080 was tested, and 31 mutants with significantly reduced pathogenicity were screened out and their biology Character analysis. Molecular validation showed that all of these low-virulence mutants were positive, and 25 T-DNAs were inserted as single copies. Compared with the wild-type strain Vd080, the 25 low-virulent mutant colonies had abundant morphological changes. In addition to the eight mycobacterium-type mutants with the same shape as the wild-type strain, three strains of yellow mycelia and two white Mycelia and 12 intermediate mutants. In addition, most of the mutants showed significant changes in growth rate, sporulation and crude toxin production, with no significant correlation between the various physiological indices. The flanking sequences of T-DNA of these 25 single-copy low-virulence mutants were further obtained by TAIL-PCR and the genomic database of Vd Ls.17. The pathogenicity of Verticillium dahliae was successfully cloned from Vd080 Force-related genes.