芸薹生链格孢（Alternaria brassicicola）AbSte5基因的克隆及功能研究

发布时间：2018-04-25 03:02

  本文选题：Alternaria + brassicicola　； 参考：《山东农业大学》2015年硕士论文

【摘要】：芸薹生链格孢(Alternaria brassicicola)引起的黑斑病是十字花科蔬菜农业生产上的一大难题,早期研究显示,Fus3-MAPK信号途径在A.brassicicola的致病性过程中起到重要的调控作用,前期在信号传导特异性方面对锚定作用已进行研究,而未对其支架蛋白作用进行详细研究。本研究主要以芸薹生链格孢菌(A.brassicicola)为对象,通过构建AbSte5基因缺失突变体与野生菌对比研究,进而针对MAPK信号转导途径中的Ste5基因功能进行研究。其主要结果如下;利用酿酒酵母saccharomyces cerevisiae Ste5基因序列在建立的A.brassicicola基因组数据库中进行本地比对,找到A.brassicicola中Ste5同源基因。根据下载的Ste5基因序列,设计特异性引物从A.brassicicola中成功扩增Ab Ste5基因以及结合RT-PCR技术扩增得到cDNA,该基因全长3327bp,cDNA3276bp,含有1个内含子且符合GT-AG法则。通过SMART蛋白质功能预测网站对A.brassicicola Ste5蛋白质序列进行分析,并与来源于其它真菌中的Ste5同源基因比对发现在序列上同源性虽低,但氨基酸序列在结构上具有极高的相似性,发现都具有RING、PH以及vWA功能域,因此推测A.brassicicola中的Ste5基因也是一种支架蛋白。构建AbSte5基因缺失突变载体pUCATPH-Ab Ste5以及M13F/M13R引物扩增新构建质粒PCR产物利用PEG介导方法转化A.brassicicola原生质体,通过PCR法及RT-PCR技术进一步验证并筛选得到了AbSte5基因缺失突变体,并通过southern杂交技术得出基因单拷贝插入缺失。在菌落表型、菌丝和分生孢子形态、黑色素以及致病性检测上将AbSte5基因缺失突变体与野生菌进行比较研究。结果发现AbSte5基因缺失突变体在PDA上生长速度下降,而且不能产生分生孢子,当采用离体叶片接种法接种未人工刺伤白菜叶片时,Ab Ste5基因缺失突变体不能侵染白菜叶片,当接种刺伤白菜叶片时,亦丧失致病力。扩增AbSte5全基因以pCB1532质粒为载体构建互补载体转化AbSte5突变菌株原生质体得到互补体后,其生物学性状等均恢复到野生菌水平。由此可以推测,AbSte5基因与菌丝形态、分生孢子的形成、致病性等有密切的关系。
[Abstract]:The black spot caused by Alternaria brassicicola (Alternaria brassicicola) is a difficult problem in the agricultural production of cruciferous vegetables. Early studies showed that Fus3-MAPK signaling pathway plays an important role in regulating the pathogenicity of A.brassicicola. The effect of anchoring on signal transduction specificity has been studied, but not on the role of scaffold protein. The purpose of this study was to study the function of Ste5 gene in MAPK signal transduction pathway by constructing AbSte5 gene deletion mutants and wild bacteria. The main results are as follows: using the sequence of saccharomyces cerevisiae Ste5 gene of Saccharomyces cerevisiae to carry out local alignment in the established A.brassicicola genomic database, the homologous gene of Ste5 in A.brassicicola was found. According to the downloadable Ste5 gene sequence, a specific primer was designed to amplify the Ab Ste5 gene from A.brassicicola and to amplify the cDNA by combining RT-PCR technique. The gene was 3327bpcDNA3276bp in length, containing an intron and conforming to the GT-AG rule. The sequence of A.brassicicola Ste5 protein was analyzed by SMART protein function prediction website, and compared with Ste5 homology gene from other fungi. Although the sequence homology was low, the amino acid sequence had very high similarity in structure. Both RINGP PH and vWA functional domains were found, so it is assumed that the Ste5 gene in A.brassicicola is also a scaffold protein. The AbSte5 gene deletion vector pUCATPH-Ab Ste5 and M13F/M13R primers were constructed to amplify the newly constructed plasmid PCR products. The A.brassicicola protoplasts were transformed into A.brassicicola protoplasts by PEG mediated method. The AbSte5 gene deletion mutants were further verified and screened by PCR and RT-PCR techniques. The single copy insertion deletion was obtained by southern hybridization. In colony phenotype, mycelium and conidial morphology, melanin and pathogenicity test, AbSte5 gene deletion mutants were compared with wild bacteria. The results showed that the growth rate of AbSte5 gene deletion mutants on PDA was decreased and conidia could not be produced. The absence of Ste5 gene mutant could not infect Chinese cabbage leaves when it was inoculated in vitro without artificial prick. When inoculated to injure cabbage leaves, it also loses pathogenicity. When the AbSte5 gene was amplified from pCB1532 plasmid and transformed into the protoplast of AbSte5 mutant strain, the biological characters were recovered to the level of wild bacteria. It can be inferred that the AbSte5 gene is closely related to hyphal morphology, conidial formation and pathogenicity.
【学位授予单位】：山东农业大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S432.44

【参考文献】

相关期刊论文 前3条

1 王洪凯,林福呈,王政逸;植物病原真菌附着胞的机械穿透力[J];菌物学报;2004年01期

2 易龙,肖崇刚,马冠华,王万能,龙良鲲;防治烟草赤星病有益内生细菌的筛选及抑菌作用[J];微生物学报;2004年01期

3 王革,周晓罡,方敦煌,李天飞;木霉拮抗烟草赤星病菌菌株的筛选及其生防机制[J];云南农业大学学报;2000年03期

，

本文编号：1799442