灰葡萄孢分生孢子产生相关基因的克隆及功能分析

摘要

[Objective] To further investigate molecular mechanism of conidiospore formation, infection and pathogenicity of Botrytis cinerea, the gene related to conidiospore formation was cloned and characterized. [Methods] A mutant with no conidiospore, named BO78, was found by screening T-DNA insertional mutant library of B. cinerea. and it was testified by PCR and Southern Blotting techniques. The flanking sequence of T-DNA insertion site was acquired by using TAIL-PCR. The sequence of the T-DNA inserted gene was obtained by scanning the B. cinerea gene bank. The mutant was further identified by PCR and RT-PCR respectively. The function of the gene was studied by analysing the colony morphology, growth rate, cell wall degrading enzyme activity, biological activity of crude toxin and pathogenicity of the mutant strain on tomato leaves as well as the expression level of genes related to pathogenicity. [Results] T-DNA insertion site was defined in the initiation codon of BC1G_127O7.1 gene, and the mutant gene was identified as BC1GJ2707.1 by RT-PCR technology. The full-length DNA sequence of BC1G12707.1 was 135 bp, and encoded a 44 amino acids hypothetical protein. Compared to the wild type strain, the mutant strain colony was white, growed slowly, did not produce conidium and sclerotia on PDA medium, but showed stronger pathogenicity on tomato leaves and cell wall degrading enzyme activity, higher expression level of genes related to pathogenicity, such as cell wall degrading enzyme gene cutA and Bcpg, genes (PKA1, PKA2, Bmp3 and Bac) involved in signal pathway, gene (BcBOT2) encoding sesquiterpene synthase, gene (Lacl) encoding melanin and transmembrane protein gene Btpl. [Conclusion] The BC1G12707.1 gene was involved in conidiation, sclerotia formation and pathogenicity in B. cinerea.%[目的]克隆灰葡萄孢分生孢子产生相关基因,并研究其功能,为进一步研究灰葡萄孢分生孢子产生机理和灰葡萄孢侵染及致病机理奠定基础.[方法]通过筛选灰葡萄孢ATMT突变体库,获得一株不能产生分生孢子的突变菌株BCt78,采用PCR和Southern Blotting技术,对突变菌株BCt78进行分子鉴定.利用TAIL-PCR技术获得T-DNA插入位点的侧翼序列;将所获得侧翼序列与灰葡萄孢基因组数据库中的已知基因序列进行BLAST分析,推测出T-DNA的插入位点;通过PCR进一步验证T-DNA的插入位点,利用RT-PCR技术确定突变基因；最后对突变菌株的菌落形态、生长速度、胞壁降解酶活力、粗毒素的生物活性、对番茄叶片的致病能力及部分致病相关基因的表达情况进行研究.[结果]TAIL-PCR结果证实T-DNA插入到灰葡萄孢BCIG 12707.1基因的ATG起始密码子区；RT-PCR结果证实突变基因为BCIG_12707.1,该基因DNA全长为135 bp,编码一个44个氨基酸的假定蛋白(Hypothetical protein).突变菌株在PDA培养基上菌落呈灰白色,生长速度减慢,不能产生分生孢子及菌核；对番茄叶片的致病性增强,且胞壁降解酶(PG、PMG和Cx)活力增强；突变菌株中参与细胞壁降解的角质酶基因cutA和多聚半乳糖醛酸酶基因Bepg1,信号转导途径基因(PKA1、PKA2、Bac、Bmp3),产毒素基因BcBOT2(Sesquiterpene synthase),漆酶基因Lac1,跨膜蛋白基因Btp1表达都增强.[结论]BC1G_ 12707.1基因在灰葡萄孢分生孢子产生、菌核形成及致病力等方面起重要作用.