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根癌农杆菌介导灰葡萄孢遗传转化及T-DNA侧翼序列分析

标题: 根癌农杆菌介导灰葡萄孢遗传转化及T-DNA侧翼序列分析
英文标题: Agrobacterium-mediated Transformation of Botrytis Cinerea and Analysis of Flanking Sequences
作者: 郑蒙
出版时间: 2009-01-01
所在大学: 河北农业大学
关键词: 灰葡萄孢,农杆菌介导的遗传转化,T-DNA插入突变,TAIL-PCR,序列分析
英文关键词: Botrytis cinerea,Agrobacterium tumefaciens–mediated transformation,T-DNA inserted mutation,TAIL-PCR,sequence analysis
论文级别: 硕士
学位: 学位论文
导师: 董金皋
专业: 植物病理学
提交时间: 2009
摘要: 灰葡萄孢(Botrytis cinerea)既是危害经济作物的重要植物病原真菌,也是研究植物与微生物相互作用的重要模式生物之一,从全基因组水平分析研究该真菌关键基因的功能,可以加速对灰葡萄孢生物学及其致病分子机制的认识,为培育持久抗病品种和制定灰霉病持续管理的策略提供理论依据.农杆菌介导(Agrobacterium tumefaciens-mediated transformation,ATMT)的遗传转化技术,以其特有的优点成为研究真菌与其寄主互作的有力工具. 本研究利用ATMT技术,创建了灰葡萄孢突变体,并对部分突变体进行了分析,具体结果如下: (1)为获得灰葡萄孢转化子,对农杆菌介导遗传转化条件进行了优化,确定了筛选转化子最适潮霉素浓度为100 μg/mL,头孢噻肟钠与羧卞青霉素对农杆菌有效抑制的浓度均为200 μg/mL.确定了共培养最佳方式是灰葡萄孢分生孢子和活化的根癌农杆菌共同涂于玻璃纸上培养,共培养的最佳时间为48 h.应用最适的转化条件,从105个分生孢子中平均获得120~150个灰葡萄孢转化子. (2)本试验已得到400多个转化子,通过继代培养初步证明插入到灰葡萄孢基因组中的潮霉素抗性基因可稳定遗传.对所获得的部分转化子进行PCR及Southern blot验证.根据T-DNA上已知潮霉素基因序列设计引物进行PCR扩增,检测结果发现所检测的转化子均含有目的潮霉素基因片段.随机选取其中6个转化子进行Southern blot验证,结果发现6个转化子均有杂交信号,且其中5个为单拷贝插入. (3)通过TAIL-PCR技术,扩增获得T-DNA插入位点的侧翼序列.对28个有效扩增片段的序列分析结果表明,19个T-DNA边界序列为灰葡萄孢序列,9个为载体主干序列.将定位的17个突变体与野生型菌株进行生物学性状比较,发现获得的部分突变体在菌株颜色、菌核的有无、分生孢子形态及产孢量方面发生明显变化.致病性测定结果表明,有1个突变体完全丧失致病能力. 本文利用农杆菌转化方法成功地将T-DNA插入灰葡萄孢基因组,获得了一批基因被标签的突变体,并发现了若干关键性状对应的基因,是灰葡萄孢功能基因组学的良好开端,具有重要的科学意义和应用前景.
英文摘要: Botrytis cinerea is not only an important plant pathogenic fungus threatening economical crop, but also a important model fungus in studying plant-microbe interaction. Dissection of gene functions at the whole genome level will speed up our understanding in the fungal biology of B.cinerea and its pathogenisis molecular mechanism, provide theoretical evidence for breeding plant with durable resistance and the disease integrated mangament. The technique of the Agrobacterium tumefaciens-mediated transformation(ATMT) has become a useful tool for studying the interaction between fungi and their hosts by its special advantage.In this thesis, a transformation system of B.cinerea mediated by ATMT had been established and several characters of some transformants have been analyzed. The results are as follows:(1) In order to obtain transformants of B.cinerea, the conditions of the Agrobacterium- mediated transformation were optimized. The result showed that 50μg/mL was the most suitable concentration of hygromycin B for screening transformants and 200μg/mL cefotaxime and carbenicillin had the best effective to inhibit Agrobacterium growth. Conidia of B. cinerea and active Agrobacterium co-smeared on glass paper was the best way of culture. 48 h was the fittest co-cultivation time. Using this optimized transformation system, about 120-150 transformants of B.cinerea were obtained from 105 spores.(2) In this study, more than 400 transformants were obtained and hygromycin resistance gene were mitotically stable after several subcultures. Parts of transformants were screened by PCR amplification and Southern blot. The primers were designed by the sequence of hph gene which we have already known. The results showed that all the transformants we screened contained the target hph fragment. Among those transformants, 6 mutants were identified by Southern blot, which have been screened by PCR. The results showed that all the 6 mutants had the blot signal and five of them contained only one copy at random site.(3) Sequence analysis of 28 fragments amplified by thermal asymmetric interlaced PCR(TAIL-PCR) showed that 19 of them were B.cinerea genomic DNA, and 9 fragments contained vector backbone sequences. Comparison between mutant strains and wild strains indicated that some biological characters were changed in some of the mutants, and the characters included colony color, sclerotia production, conidium morphology, and sporulation. Pathogenicity results showed that only a singal mutant lost virulence.In summary, T-DNA was successfully inserted into genome of the B.cinerea by ATMT technique; a large scale of T-DNA insertional mutants were generated; and several genes related to important biological functions were found to be tagged by T-DNA in this study. This is a good start for functional genomics of the fungus and it will have an important impact on science and future application.