芸薹生链格孢AbPbs2基因功能及下游MAPK锚定作用位点的研究

发布时间：2018-08-10 20:54

【摘要】：芸薹生链格孢(Alternaria brassicae)是一种在自然界中分布十分广泛的真菌,其寄主范围广,对环境适应性强,既是重要植物病原菌又是常见的腐生菌,由其引起的十字花科黑斑病常造成严重的经济损失。研究发现其高渗透压甘油信号转导途径在孢子生长、渗透胁迫、抗药性和致病性等方面起着重要的调控作用。本研究拟进一步深入研究该信号途径激酶MAPK(AbHog1)和MAPKK(AbPbs2)之间的锚定反应机制,明确二者身上存在的锚定作用位点,全面认识芸薹生链格孢高渗透压甘油信号转导途径的作用机理,深入了解该菌致病性的分子作用机理,为未来开发以该途径为靶标的新型杀菌剂提供重要的理论和技术支持。本文主要结果如下:(1)利用已知芸薹生链格孢Pbs2基因序列以及生物信息学软件分析后设计其特异性引物,通过PCR的方法扩增获得AbPbs2基因编码区DNA。经过Blast比对证实与GenBank中已有的基因同源性达到97.98%。分析其蛋白质序列后证实了成功获得AbPbs2基因。真菌HOG途径的研究不断深入,了解其代谢途径的各种相关因子的作用机理及代谢机制,能够为人类充分认识与利用微生物资源奠定理论基础。芸薹生链格孢(A.brassicicola)AbPbs2基因的克隆及功能鉴定,为进一步研究真菌生长、致病性、环境适应性等方面奠定了重要基础。(2)通过原生质体转化体系构建了AbPbs2的基因插入突变体,对比了△AbPbs2和野生菌在生长速度、产孢能力、渗透压敏感性、杀菌剂咯菌腈抗性和致病性方面的差异。结果显示菌丝生长变化不大、但产孢能力受到严重损伤,且对渗透压的敏感性增强,此外病原菌致病性也较大程度丧失。这些结果表明AbPbs2基因参与病原菌产孢、渗透压调节和致病性调控。(3)采用重叠延伸PCR的方法,扩增得到了D位点缺失的AbPbs2△D,通过原生质体转化体系成功构建了△AbPbs2/AbPbs2互补体和△AbPbs2/AbPbs2△D互补体,并将两者在生长速度、产孢能力、渗透压敏感性、杀菌剂咯菌腈抗性和致病性方面进行比对,结果显示D位点缺失的AbPbs2互补△AbPbs2突变体后,互补体的功能均没有恢复到野生菌的性状,故推测D位点在AbPbs2与AbHog1的锚定作用中起着重要作用。
[Abstract]:Alternaria brassica (Alternaria brassicae) is a widely distributed fungus in nature, its host range is wide, and it has strong adaptability to environment. The cruciferous black spot caused by it often causes serious economic losses. It was found that high osmotic glycerol signaling pathway plays an important role in spores growth, osmotic stress, drug resistance and pathogenicity. The aim of this study was to further study the anchoring reaction mechanism between MAPK (AbHog1) and MAPKK (AbPbs2), to identify the anchoring sites in the two signaling pathways, and to fully understand the mechanism of the signal transduction pathway of high osmotic glycerol in Alternaria brassica. In order to provide important theoretical and technical support for the future development of new fungicides targeted at this pathway, the molecular mechanism of pathogenicity of this bacterium is deeply understood. The main results are as follows: (1) the specific primers were designed by using known Pbs2 gene sequence of Alternaria brassica and bioinformatics software. The coding region of AbPbs2 gene was amplified by PCR. The homology of GenBank gene with Blast was 97.98%. The AbPbs2 gene was successfully obtained by analyzing its protein sequence. The study of fungal HOG pathway has been further studied. Understanding the action mechanism and metabolic mechanism of various factors related to its metabolic pathway can lay a theoretical foundation for human beings to fully understand and utilize microbial resources. The cloning and functional identification of the AbPbs2 gene of A.brassicicola in Brassica brassica have laid an important foundation for the further study of fungal growth, pathogenicity and environmental adaptability. (2) the gene insertion mutant of AbPbs2 was constructed through protoplast transformation system. The differences of growth rate, sporulation ability, osmotic sensitivity, fungicides resistance and pathogenicity between AbPbs2 and wild bacteria were compared. The results showed that the mycelium growth had little change, but the sporulation ability was seriously damaged, the sensitivity to osmotic pressure was enhanced, and the pathogenicity of the pathogen was also largely lost. These results suggest that AbPbs2 gene is involved in sporulation, osmotic pressure regulation and pathogenicity regulation of pathogenic bacteria. (3) overlapping extended PCR was used. AbPbs2 D with D locus deletion was obtained by amplification. AbPbs2/AbPbs2 complement and AbPbs2/AbPbs2 D complement were successfully constructed by protoplast transformation system, and the growth rate, sporulation ability and osmotic pressure sensitivity were determined. The results of comparison of fungicides and pathogenicity showed that the function of AbPbs2 complementary AbPbs2 mutants without D locus was not restored to wild bacteria. It is inferred that D site plays an important role in the anchoring of AbPbs2 and AbHog1.
【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

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