禾谷镰刀菌磷脂酶D基因家族功能分析

发布时间：2018-05-14 12:51

本文选题：禾谷镰刀菌 + FgPLD　；参考：《山东农业大学》2017年硕士论文

【摘要】：小麦赤霉病(Fusarium head blight,FHB)是禾谷类作物上的重要病害,而禾谷镰刀菌(Fusarium graminearum)是赤霉病的主要病原菌。磷脂酶D(phospholipase D,PLD)是重要的磷脂水解酶,在真核生物多种生理过程中发挥关键作用。目前对于植物病原真菌PLD的功能研究还未有报道。本研究从禾谷镰刀菌基因组中鉴定出3个Fg PLD基因,经Split-marker PCR技术构建禾谷镰刀菌Fg PLD敲除片段,利用同源重组原理和原生质体转化法,获得3个Fg PLDs基因敲除突变体?plds,并获得Fg PLD1的回复菌株?pld1-C。为阐明Fg PLD在禾谷镰刀菌生长发育和致病性中的功能,进一步研究Fg PLD基因敲除突变体的相关表型变化。敲除Fg PLD1(?pld1)影响菌丝生长,分生孢子产生和子囊壳形成等。与野生型菌株相比,?pld1突变体的菌落直径减小了约58.23%;?pld1突变体菌落形态还表现出较短的气生菌丝和更多的菌丝分枝。与野生型菌株相比,?pld1突变体的分生孢子产量减少约68.71%;在YEPD培养基中孵育6 h,约64.72%的?pld1分生孢子萌发,表明Fg PLD1在分生孢子产量和孢子萌发方面发挥重要功能。?pld1突变体不能形成子囊壳,表明其在有性生殖方面存在缺陷。?pld1突变体病情指数显示其毒力约降低95%;与野生型菌株相比,?pld1突变体感染小麦种子中的DON产量显著降低;对TRI5和TRI6的表达水平的进一步分析,显示?pld1突变体中TRI5和TRI6的相对表达水平显着低于野生型菌株。而?pld2和?pld3突变体在菌落生长、无性和有性生殖以及致病性等方面的表型,与野生型菌株差别均不明显。回复菌株?pld1-C在菌落生长、无性和有性生殖以及致病性等方面的表型均回复到了野生型菌株的水平。为了解析Fg PLD1-PX结构域的功能,对Fg PLD1-PX区单独敲除,突变体命名?pld1px。结果显示?pld1px与?pld1具有相似的表型,包括菌丝生长、分生孢子产量和致病性等,表明PX结构域对于Fg PLD1功能的发挥是重要的。总之,这些结果表明,在F.graminearum中Fg PLD1对于菌丝生长、侵染能力以及无性和有性生殖等是必要的。而Fg PLD2和Fg PLD3基因在调控菌体生长发育以及致病性等方面的作用不显著。另外还证明PX结构域是影响Fg PLD1功能的关键区域。
[Abstract]:Fusarium head blightFHB) is an important disease in cereal crops, while Fusarium graminearum is the main pathogen of Fusarium graminearum. Phospholipase (D(phospholipase) is an important phospholipid hydrolase, which plays a key role in many physiological processes of eukaryotes. At present, the study on the function of plant pathogenic fungi PLD has not been reported. In this study, three FG PLD genes were identified from Fusarium graminearum genomes. FG PLD knockout fragments were constructed by Split-marker PCR technique, and homologous recombination principle and protoplast transformation method were used to construct FG PLD knockout fragments from Fusarium graminearum. Three FG PLDs knockout mutants were obtained, and the response strain of FG PLD1 was obtained. In order to elucidate the function of FG PLD in the growth, development and pathogenicity of Fusarium graminearum, the phenotypic changes of FG PLD knockout mutants were further studied. Knock out FG PLD 1 pld 1) affect hyphal growth, conidium production and cotyledon formation. Compared with wild-type strain, the colony diameter of the mutant was reduced about 58.23 and the colony morphology of the mutant was also shorter than that of wild-type strain. It also showed shorter hypha and more branching of hyphae. Compared with wild-type strain, the conidial yield of the mutant was reduced by 68.71%, and the conidial germination was about 64.72% in YEPD medium for 6 h. The results showed that FG PLD1 played an important role in conidial yield and spore germination. The results showed that the disease index of the mutant showed that its virulence was about 95% lower than that of wild-type strain, the DON yield in wheat seeds was significantly lower than that of wild-type strain, and the expression levels of TRI5 and TRI6 were further analyzed. The results showed that the relative expression level of TRI5 and TRI6 in the mutant was significantly lower than that in the wild-type strain. But the phenotypes of the mutants, such as colony growth, asexual, sexual reproduction and pathogenicity, were not significantly different from those of wild-type strains. The phenotypes of pld1-C in colony growth, asexual, sexual reproduction and pathogenicity were all recovered to the level of wild-type strains. In order to analyze the function of FG PLD1-PX domain, the FG PLD1-PX domain was knocked out alone, and the mutant was named pld1px. The results showed that the phenotypes of pld1px and pld1 were similar, including mycelium growth, conidial yield and pathogenicity, which indicated that PX domain was important for the function of FG PLD1. In conclusion, these results indicate that FG PLD1 is necessary for mycelium growth, infection ability and asexual and sexual reproduction in F.graminearum. However, FG PLD2 and FG PLD3 genes did not play a significant role in regulating the growth, development and pathogenicity of bacteria. It is also proved that the PX domain is the key area affecting the function of FG PLD1.
【学位授予单位】：山东农业大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S435.121.45

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