论文部分内容阅读
【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SouthernBlotting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BC1G_12707.1基因的ATG起始密码子区;RT-PCR结果证实突变基因为BC1G_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Bcpg1,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。
【Objective】 Cloning the genes related to conidia production of Botrytis cinerea and studying its function, which laid the foundation for further study on the mechanism of conidia production and the infection and pathogenic mechanism of Botrytis cinerea. 【Method】 A mutant strain BCt78 that could not produce conidia was obtained by screening the ATMT mutant library of Botrytis cinerea, and the mutant BCt78 was identified by PCR and SouthernBlotting. TAIL-PCR technique was used to obtain the flanking sequence of T-DNA insertion site. BLAST analysis of the obtained flanking sequence and the known gene sequence in the genome database of Botrytis cinerea predicted the insertion site of T-DNA. Finally, the colony morphology, growth rate, cell wall degrading enzyme activity, biological activity of crude toxin, the pathogenicity of tomato leaves and part of the mutant strains Pathogenicity-related genes were studied. 【Result】 The results of TAIL-PCR confirmed that the T-DNA was inserted into the ATG start codon region of BC1G_12707.1 gene. The result of RT-PCR confirmed that the mutant gene was BC1G_12707.1. The full- A 44 amino acid hypothetical protein. The mutant strains showed gray-white colonies on PDA medium, the growth rate was slow, conidia and sclerotia could not be produced, the pathogenicity to tomato leaves was enhanced, and the activity of cell-wall degrading enzymes (PG, PMG and Cx) The cuta and polygalacturonase genes Bcpg1, signal transduction pathway genes (PKA1, PKA2, Bac, Bmp3), BcBOT2 (Sesquiterpene synthase), laccase Lac1, Transmembrane protein gene Btp1 expression are enhanced. 【Conclusion】 The BC1G_12707.1 gene plays an important role in conidial generation, sclerotia formation and virulence of Botrytis cinerea.