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灰葡萄孢T-DNA插入突变体的筛选及分析

标题: 灰葡萄孢T-DNA插入突变体的筛选及分析
英文标题: The Screening and Characterization of Botrytis Cinerea Mutants Generated by T-DNA Insertion
作者: 张娇
出版时间: 2010-01-01
所在大学: 河北农业大学
关键词: 灰葡萄孢,TAIL-PCR,T-DNA插入突变,致病性
英文关键词: Botrytis cinerea,TAIL-PCR,T-DNA insertional mutagensis,Pathogenicity
论文级别: 硕士
学位: 学位论文
导师: 韩建民%董金皋
专业: 植物学
提交时间: 2010
摘要: 通过对农杆菌介导转化灰葡萄孢(Botrytis cinerea)获得的转化子进行筛选,得到一株不形成菌核,孢子产量明显减少,对番茄叶片和果实致病性明显增强的菌株BCt41.通过对该菌株进行PCR、Southern杂交鉴定,证明该菌株为遗传稳定的单拷贝T-DNA插入突变体.通过TAIL-PCR技术获得了T-DNA插入位点左翼264 bp的DNA序列,与灰葡萄孢数据库比对表明:T-DNA插入位点位于基因BC1G02176.1的外显子2中.该基因编码区长1206 bp,含一个54 bp内含子,编码383个氨基酸,基因功能未知.本研究结果为进一步研究该基因在灰葡萄孢分生孢子发育、菌核形成及致病过程中的作用奠定了基础.
英文摘要: The transformant BCt41 was found by screening the transformants of Botrytis cinerea produced by Agrobacterium tumefaciens-mediated method, which lost the ability of producing sclerotia, and significantly reduced spores production on PDA medium, but showed the stronger pathogenicity to tomato leaves and fruits compared to the wild type strain. PCR amplification and Southern blotting hybridization on the genomic DNA of BCt41 showed that the genomic DNA of BCt41had carried steady heritable single copy T-DNA. The 264 bp flanking sequence of T-DNA insertion sites was gained by amplifing genomic DNA of BCt41 using thermal asymmetric interlaced PCR (TAIL-PCR) technique. Comparison of 264 bp sequence with Botrytis cinerea database showed that T-DNA insertion sites located in exon 2 of the gene BC1G02176.1. The 1206 bp coding region of BC1G02176.1 gene with a 54 bp intron, encoded 383 amino acid, and the function of BC1G02176.1 gene was unknown. Our results lay foundations for further studying BC1G02176.1 gene functions in Botrytis cinerea involving the development of conidia and the formation of sclerotia as well as pathogenicity to tomato leaves and fruits.