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为获得灰葡萄孢的致病相关基因并研究其基因功能,筛选灰葡萄孢的T-DNA插入突变体库,获得了一株致病力增强的突变体BCt98。利用PCR和Southern Blotting技术,对突变体BCt98进行鉴定。利用TAIL-PCR技术结合生物信息学方法,确定了突变体BCt98中T-DNA插入位点位于BC1G_07014.1基因的第3个外显子上。利用RT-PCR技术,确定了突变体BCt98的突变基因为BC1G_07014.1。突变体BCt98生长速度较快,菌落颜色较浅,菌丝较为致密,不产生分生孢子和菌核,且胞壁降解酶(PMG、PG和Cx)及毒素活性较野生型明显增强。表明BC1G_07014.1基因在灰葡萄孢生长、发育和致病力调控方面发挥重要作用,且参与调控病菌的胞壁降解酶活性和毒素活性。
In order to obtain the pathogenicity-related genes of Botrytis cinerea and to study its gene function, a mutant strain BCt98 with enhanced pathogenicity was obtained by screening T-DNA insertion mutant library of Botrytis cinerea. The mutant BCt98 was identified by PCR and Southern Blotting. Using TAIL-PCR combined with bioinformatics methods, it was confirmed that the T-DNA insertion site of BCt98 was located on the third exon of BC1G_07014.1 gene. Using RT-PCR, the mutant BCt98 gene was identified as BC1G_07014.1. The mutant BCt98 grew faster, the color of the colony was lighter, the mycelia were more dense, the conidia and sclerotia were not produced, and the activities of cell wall degrading enzymes (PMG, PG and Cx) and toxins were significantly enhanced compared with the wild type. The results indicated that BC1G_07014.1 gene plays an important role in the regulation of the growth, development and pathogenicity of Botrytis cinerea, and is involved in the regulation of cell wall degrading enzymes activity and toxin activity.