【摘要】：蕓薹生鏈格孢(Alternaria brassicae)是一種在自然界中分布十分廣泛的真菌,其寄主范圍廣,對環(huán)境適應性強,既是重要植物病原菌又是常見的腐生菌,由其引起的十字花科黑斑病常造成嚴重的經濟損失。研究發(fā)現(xiàn)其高滲透壓甘油信號轉導途徑在孢子生長、滲透脅迫、抗藥性和致病性等方面起著重要的調控作用。本研究擬進一步深入研究該信號途徑激酶MAPK(AbHog1)和MAPKK(AbPbs2)之間的錨定反應機制,明確二者身上存在的錨定作用位點,全面認識蕓薹生鏈格孢高滲透壓甘油信號轉導途徑的作用機理,深入了解該菌致病性的分子作用機理,為未來開發(fā)以該途徑為靶標的新型殺菌劑提供重要的理論和技術支持。本文主要結果如下:(1)利用已知蕓薹生鏈格孢Pbs2基因序列以及生物信息學軟件分析后設計其特異性引物,通過PCR的方法擴增獲得AbPbs2基因編碼區(qū)DNA。經過Blast比對證實與GenBank中已有的基因同源性達到97.98%。分析其蛋白質序列后證實了成功獲得AbPbs2基因。真菌HOG途徑的研究不斷深入,了解其代謝途徑的各種相關因子的作用機理及代謝機制,能夠為人類充分認識與利用微生物資源奠定理論基礎。蕓薹生鏈格孢(A.brassicicola)AbPbs2基因的克隆及功能鑒定,為進一步研究真菌生長、致病性、環(huán)境適應性等方面奠定了重要基礎。(2)通過原生質體轉化體系構建了AbPbs2的基因插入突變體,對比了△AbPbs2和野生菌在生長速度、產孢能力、滲透壓敏感性、殺菌劑咯菌腈抗性和致病性方面的差異。結果顯示菌絲生長變化不大、但產孢能力受到嚴重損傷,且對滲透壓的敏感性增強,此外病原菌致病性也較大程度喪失。這些結果表明AbPbs2基因參與病原菌產孢、滲透壓調節(jié)和致病性調控。(3)采用重疊延伸PCR的方法,擴增得到了D位點缺失的AbPbs2△D,通過原生質體轉化體系成功構建了△AbPbs2/AbPbs2互補體和△AbPbs2/AbPbs2△D互補體,并將兩者在生長速度、產孢能力、滲透壓敏感性、殺菌劑咯菌腈抗性和致病性方面進行比對,結果顯示D位點缺失的AbPbs2互補△AbPbs2突變體后,互補體的功能均沒有恢復到野生菌的性狀,故推測D位點在AbPbs2與AbHog1的錨定作用中起著重要作用。
[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.
【學位授予單位】：山東農業(yè)大學
【學位級別】：碩士
【學位授予年份】：2015
【分類號】：S432.44

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