灰葡萄孢中产孢相关基因的功能研究

发布时间：2018-06-06 01:32

本文选题：灰葡萄孢 + 分生孢子　；参考：《华中农业大学》2017年硕士论文

【摘要】：灰葡萄孢是一种气传性植物病原真菌,它能够侵染200多种植物,包括番茄、草莓、葡萄以及多种花卉。灰葡萄孢的抗逆性极强,可靠菌核或分生孢子度过恶劣环境。研究灰葡萄孢菌核发育、分生孢子产生或致病机理可为有效防治灰霉病奠定理论基础。本研究以灰葡萄孢菌丝生长时期和菌核发育时期的基因表达谱为基础,挑选出了2个在这两个阶段基因表达量差异较大的基因BC1G_00689和BC1G_03293,并初步研究了它们的功能,取得了以下研究结果:1.RNA-seq结果表明基因BC1G_00689在菌核发育时期表达下调,表达量是菌丝生长时期的18%,该基因编码一个包含酸性鞘磷脂酶结构域的蛋白质。基因BC1G_03293在菌核发育时期表达上调,表达量比菌丝生长时期上调了78倍。该基因编码的蛋白质与大肠杆菌中周质空间麦芽糖结合蛋白的结构相似度较高,目前在真菌中还没有相关蛋白质功能的报道。为了研究基因BC1G_00689的功能,我们通过同源重组的方法得到了敲除转化子△BC1G_00689-19和△BC1G_00689-21。也得到了基因BC1G_03293的敲除转化子△BC1G_03293-2和△BC1G_03293-4,并运用基因互补技术得到了互补转化子△BC1G_03293-2-C2和△BC1G_03293-2-C3。2.敲除转化子△BC1G_00689-19和△BC1G_00689-21的菌落形态、菌丝尖端形态、菌丝生长速度、对番茄叶片的致病力以及菌核干重与野生菌株B05.10相比无明显差异,但敲除转化子的产孢量明显下降,约为出发菌株B05.10产孢量的70%。结果说明BC1G_00689基因在灰葡萄孢产孢过程中可能发挥重要作用。3.BC1G_03293基因敲除后产孢量明显下降,大约为出发菌株B05.10产孢量的45%,并且BC1G_03293基因互补可使敲除转化子的产孢量得到明显恢复。在加麦芽糖和蔗糖的基本培养基上敲除转化子的菌丝生长速度、菌落形态以及菌核干重与B05.10相比无明显区别。结果说明BC1G_03293基因参与调控了灰葡萄孢的分生孢子产生。
[Abstract]:Grapevine spore is an airborne plant pathogen that can infect more than 200 species of plants, including tomatoes, strawberries, grapes and many flowers. Grapevine spores have strong resistance to stress and reliable sclerotia or conidial spore through the harsh environment. Studies on the development of sclerotia of grapevine and the mechanism of conidial production or pathogenesis may lay a theoretical foundation for the effective control of grey mold. Based on the gene expression profiles of hyphal growth and sclerotia development, two genes, BC1G_00689 and BC1G03293, were selected and their functions were preliminarily studied. The following results were obtained: 1. RNA-seq results showed that the expression of BC1G_00689 gene was down-regulated during sclerotia development, and the expression level was 18 ~ (th) of mycelium growth stage. The gene encodes a protein containing acidic sphingolipase domain. The expression of BC1G_03293 was up-regulated during sclerotia development and was 78 times higher than that in hyphal growth. The protein encoded by this gene has high structural similarity to that of periplasmic space maltose binding protein in Escherichia coli, and there is no report on the function of the protein in fungi at present. In order to study the function of gene BC1G_00689, we obtained the knockout transformants BC1G_00689-19 and BC1GStack00689-21by homologous recombination. The knockout transformants BC1G_03293-2 and BC1GV03293-4 of gene BC1G_03293 were also obtained, and the complementary transformants BC1G_03293-2-C2 and BC1G03293-2-C3.2 were obtained by gene complementation technique. The colony morphology, hyphae tip morphology, mycelium growth rate, pathogenicity of tomato leaves and sclerotia dry weight of knockout transformants BC1G_00689-19 and BC1G_00689-21 were not significantly different from those of wild strain B05.10, but the sporulation of knockout transformants decreased significantly. It is about 70% of the sporulation of the original strain B05.10. The results showed that BC1G_00689 gene may play an important role in the sporulation process of grapevine spore. 3.BC1G03293 gene knockout significantly decreased the sporulation production, which was about 45% of the original strain B05.10 sporulation, and BC1G_03293 gene complement could obviously restore the sporulation of knockout transformants. The mycelium growth rate, colony morphology and sclerotia dry weight of the transformants on the basic medium supplemented with maltose and sucrose were not significantly different from those of B05.10. The results showed that BC1G_03293 gene was involved in the conidial production of grapevine.
【学位授予单位】：华中农业大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S432.44

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