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灰葡萄孢分生孢子产生能力丧失突变体筛选及相关基因功能分析

标题: 灰葡萄孢分生孢子产生能力丧失突变体筛选及相关基因功能分析
英文标题: Screening of Botrytis cinerea sporulating-defect mutants and functional analysis of sporulating-related genes
作者: 夏中博
出版时间: 2011-01-01
所在大学: 河北农业大学
关键词: 灰葡萄孢,T-DNA插入突变体,TAIL-PCR,孢子发育
英文关键词: Botrytis cinerea,T-DNA insertional mutant,TAIL-PCR,conidium development
论文级别: 硕士
学位: 学位论文
导师: 韩建民%董金皋
专业: 发育生物学
提交时间: 2011
摘要: 通过筛选灰葡萄孢ATMT突变体库,获得了2株不产生分生孢子的突变菌株--BMH174和BMH179.利用TAIL-PCR技术获得其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,突变菌株BMH174的T-DNA插入位点位于灰葡萄孢BC1G_06945.1基因第2个外显子上,通过PCR进一步验证T-DNA的插入位点.RT-PCR技术确定了突变基因为BC1G_06945.1.生物信学分析发现,BC1G_06945.1基因全长2595 bp,含有4个内含子,编码865个氨基酸,基因产物功能未知,该基因与核盘菌SS1G_02042基因的同源性达82.3%.突变菌株BMH179的T-DNA插入位点位于灰葡萄孢BC1G_02800.1基因第二个外显子上,通过PCR证实了T-DNA的插入位点.RT-PCR技术确定了突变基因为BC1G_02799.1.生物信学分析发现,该基因DNA全长为1951 bp,含有2个内含子,编码615个氨基酸,该基因编码的蛋白与ABC转运蛋白高度同源,同源性达98%.研究发现,突变菌株BMH174和突变菌株BMH179的菌落均呈白色、生长速度均减慢,都不产生菌核,纤维素酶、果胶酶等胞壁降解酶活性都增强,对番茄叶片和果实的致病性和产毒素能力均都增强.本研究结果将为灰葡萄孢分生孢子发育、菌核形成及致病性调控的分子机理奠定基础.??
英文摘要: Two sporulating-defect mutants of Botrytis cinerea, BMH174 and BMH179, were obtained by Agrobacterium tumefaciens-mediated method. The flanking sequence of T-DNA insertion site in BMH174 was acquired by TAIL-PCR technology then the T-DNA insertion in the second exon of BC1G_06945.1 confirmed by BLAST between the flanking sequence and the known sequence in the B.cinerea gene database. The mutant gene was identified BC1G_06945.1 gene by RT-PCR. The DNA full-length sequence of BC1G_06945.1 was 2595 bp and contained 4 introns, encoded a 865 amino acids putative protein, and the function of BC1G_06945.1 gene was unknown.The homology of BC1G_06945.1 gene and Sclerotinia SS1G_02042 gene was 82.3%. The flanking sequence of T-DNA insertion site in BMH179 was acquired by TAIL-PCR technology and then the T-DNA insertion in the second exon of BC1G_02800.1 confirmed by BLAST between the flanking sequence and the known sequence in the B. cinerea gene database. The mutant gene was identified as BC1G_02799.1 located in the upstream of BC1G_02800.1 gene by RT-PCR. The DNA full-length sequence of BC1G_02799.1 was 1951 bp and contained 2 introns, encoded a 615 amino acids putative protein similar to ABC-transporter. The homology was 98%. Phenotype analysis of the mutants BMH174 and BMH179 found that the mutant strain colony were white, growed slowly, did not produce conidium and sclerotia on PDA medium, but showed the stronger pathogenicity to tomato leaves and friuts and successfully increased enzyme activity related to pathogenicity compared to the wild type strain. The results suggested that the BC1G_06945.1 and BC1G_02799.1 genes were involved in the conidium development, the sclerotia formation and pathogenicity in B. cinerea which will faciliate to understant molecular mechanism of conidium development, sclerotia formation and pathogenic in B. cinerea.??