本文選題：灰葡萄孢 + 分生孢子��； 參考：《華中農(nóng)業(yè)大學(xué)》2017年碩士論文

【摘要】：灰葡萄孢是一種氣傳性植物病原真菌,它能夠侵染200多種植物,包括番茄、草莓、葡萄以及多種花卉�；移咸焰叩目鼓嫘詷O強(qiáng),可靠菌核或分生孢子度過(guò)惡劣環(huán)境。研究灰葡萄孢菌核發(fā)育、分生孢子產(chǎn)生或致病機(jī)理可為有效防治灰霉病奠定理論基礎(chǔ)。本研究以灰葡萄孢菌絲生長(zhǎng)時(shí)期和菌核發(fā)育時(shí)期的基因表達(dá)譜為基礎(chǔ),挑選出了2個(gè)在這兩個(gè)階段基因表達(dá)量差異較大的基因BC1G_00689和BC1G_03293,并初步研究了它們的功能,取得了以下研究結(jié)果:1.RNA-seq結(jié)果表明基因BC1G_00689在菌核發(fā)育時(shí)期表達(dá)下調(diào),表達(dá)量是菌絲生長(zhǎng)時(shí)期的18%,該基因編碼一個(gè)包含酸性鞘磷脂酶結(jié)構(gòu)域的蛋白質(zhì)�；駼C1G_03293在菌核發(fā)育時(shí)期表達(dá)上調(diào),表達(dá)量比菌絲生長(zhǎng)時(shí)期上調(diào)了78倍。該基因編碼的蛋白質(zhì)與大腸桿菌中周質(zhì)空間麥芽糖結(jié)合蛋白的結(jié)構(gòu)相似度較高,目前在真菌中還沒有相關(guān)蛋白質(zhì)功能的報(bào)道。為了研究基因BC1G_00689的功能,我們通過(guò)同源重組的方法得到了敲除轉(zhuǎn)化子△BC1G_00689-19和△BC1G_00689-21。也得到了基因BC1G_03293的敲除轉(zhuǎn)化子△BC1G_03293-2和△BC1G_03293-4,并運(yùn)用基因互補(bǔ)技術(shù)得到了互補(bǔ)轉(zhuǎn)化子△BC1G_03293-2-C2和△BC1G_03293-2-C3。2.敲除轉(zhuǎn)化子△BC1G_00689-19和△BC1G_00689-21的菌落形態(tài)、菌絲尖端形態(tài)、菌絲生長(zhǎng)速度、對(duì)番茄葉片的致病力以及菌核干重與野生菌株B05.10相比無(wú)明顯差異,但敲除轉(zhuǎn)化子的產(chǎn)孢量明顯下降,約為出發(fā)菌株B05.10產(chǎn)孢量的70%。結(jié)果說(shuō)明BC1G_00689基因在灰葡萄孢產(chǎn)孢過(guò)程中可能發(fā)揮重要作用。3.BC1G_03293基因敲除后產(chǎn)孢量明顯下降,大約為出發(fā)菌株B05.10產(chǎn)孢量的45%,并且BC1G_03293基因互補(bǔ)可使敲除轉(zhuǎn)化子的產(chǎn)孢量得到明顯恢復(fù)。在加麥芽糖和蔗糖的基本培養(yǎng)基上敲除轉(zhuǎn)化子的菌絲生長(zhǎng)速度、菌落形態(tài)以及菌核干重與B05.10相比無(wú)明顯區(qū)別。結(jié)果說(shuō)明BC1G_03293基因參與調(diào)控了灰葡萄孢的分生孢子產(chǎn)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.
【學(xué)位授予單位】：華中農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：S432.44

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