ALPHA-葡萄糖苷酶家族基因在稻瘟病菌中的功能研究

发布时间：2018-04-20 00:38

本文选题：稻瘟病菌 + N-连接糖基化　；参考：《南京农业大学》2016年硕士论文

【摘要】：由稻瘟病菌(Magnaporthe oryzae)引起的水稻稻瘟病是水稻的重要病害,在世界各稻区均有发生。在适宜的环境条件下该病可迅速地蔓延扩散,能在短时间内造成大面积水稻受害,严重时减产可超过50%,甚至绝收。稻瘟病菌侵染水稻主要依靠分生孢子萌发产生芽管,芽管萌发形成附着胞,附着胞内黑色素产生巨大膨压迫使侵入钉穿透水稻表皮,侵入到植物细胞内部,从而引起病害。目前对该病的防治方法是以选育抗病品种为主,药剂防治为辅。但是由于该病原菌小种的易变性,仅依靠选育抗病品种不能达到预期效果。水稻和稻瘟病菌全基因组序列的公布对研究稻瘟病菌的致病机理和与水稻的互作模式提供了良好的条件,推动了致病相关基因的功能研究,为挖掘新型药剂靶标和制定病害防控策略提供了重要的理论依据。N-连接糖基化(N-linked glycosylation)是在内质网中对新合成的蛋白质进行糖基化修饰的一种方式。这种修饰作用首先要给新生多肽链的天冬酰胺添加一个核心寡聚糖,然后逐步切除寡聚糖的三分子葡萄糖和一分子甘露糖,形成具有相似糖链的糖蛋白。这一系列有序的加工和修饰过程有利于蛋白质抵抗消化酶、信号传导以及正确折叠。糖苷水解酶(glycosidehydrolase)是所有催化糖苷和寡糖中糖苷键水解的酶的总称,在糖蛋白N-糖基化修饰和碳水化合物降解的过程中是必不可少的。本研究在稻瘟病菌中鉴定了 N-连接糖基化过程中三个糖苷水解酶蛋白MoGlsl、MoGls2和MoGtb1并对这些蛋白编码基因的生物学功能进行了研究。采用基因敲除的方法获得了敲除突变体ΔMoGls1、AMoGls2和ΔMoGtb1。通过试验研究发现MoGLS1缺失使稻瘟病菌的有性生殖能力丧失;MoGTB1缺失使稻瘟病菌的营养生长速度减慢、产孢量显著上升;MoGLS2缺失使稻瘟病菌营养生长速度减慢,产孢量显著上升,附着胞形成延迟,有性生殖能力丧失。致病性测定表明ΔMogls2突变体的致病能力和侵染菌丝扩展能力均显著下降。进一步研究发现,ΔMogls2突变体对外界盐胁迫和渗透胁迫敏感性降低,对细胞壁胁迫敏感性升高。除此之外,ΔMogls2突变体还表现出细胞壁完整性缺陷。上述结果表明,MoGls2是稻瘟病菌生长发育过程中一个非常重要的蛋白,参与调控病菌的无性生殖、有性生殖、外界胁迫的应答、细胞壁的完整性以及侵染菌丝的扩展。
[Abstract]:Rice blast caused by Magnaporthe oryzae is an important disease of rice, which occurs in every rice area of the world. Under suitable environmental conditions, the disease can spread rapidly and spread rapidly, which can cause a large area of rice damage in a short period of time, and in severe cases, the yield can exceed 50%, or even close to the end of harvest. The germinating tubules of rice infected by Magnaporthe grisea mainly depend on conidial germination to form appressorium, and the huge swelling of melanin in attachment cells forces the invading nail to penetrate the epidermis of rice and invade into the plant cells, thus causing disease. At present, the prevention and treatment of the disease is mainly by breeding resistant varieties, supplemented by insecticide control. However, due to the variability of the pathogen race, it can not achieve the expected effect only by breeding resistant varieties. The publication of the whole genome sequence of rice and rice blast fungus provides a good condition for studying the pathogenic mechanism and interaction model of rice blast fungus, and promotes the functional study of pathogenicity related genes. It provides an important theoretical basis for digging new drug targets and formulating disease control strategies. N- linked glycosylation is a way to glycosylation of newly synthesized proteins in the endoplasmic reticulum. This modification first adds a core oligosaccharide to the asparagine of the new polypeptide chain and then gradually removes the oligosaccharide trimolecular glucose and one molecule mannose to form a glycoprotein with similar sugar chain. This sequence of ordered processing and modification is conducive to protein resistance to digestive enzymes, signal transduction, and proper folding. Glycoside hydrolase (glycoside hydrolase), which catalyzes the hydrolysis of glycoside bonds in glycosides and oligosaccharides, is essential in the process of N-glycosylation modification and carbohydrate degradation. In this study, three glycoside hydrolase proteins MoGlslMoGls2 and MoGtb1 were identified in rice blast fungus and their biological functions were studied. The mutant 螖 MoGls1AMoGls2 and 螖 MoGtb1 were obtained by gene knockout. The results showed that the loss of sexual reproduction ability of rice blast fungus caused by MoGLS1 deficiency slowed down the vegetative growth rate of M. grisea, increased the sporulation quantity significantly, and slowed down the vegetative growth rate and the sporulation quantity of M. grisea fungus by the absence of MoGLS2, which led to the decrease of vegetative growth rate and the increase of sporulation quantity of M. grisea. The formation of appura was delayed and sexual reproduction was lost. Pathogenicity test showed that the pathogenicity and hyphal expansion ability of 螖 Mogls2 mutants were significantly decreased. Further studies showed that the sensitivity of 螖 Mogls2 mutants to external salt stress and osmotic stress decreased, but to cell wall stress increased. In addition, 螖 Mogls2 mutants also showed cell wall integrity defects. These results suggest that MoGlS2 is a very important protein in the growth and development of Magnaporthe oryzae, which is involved in the regulation of asexual reproduction, sexual reproduction, the response to external stress, the integrity of cell wall and the expansion of infection hyphae.
【学位授予单位】：南京农业大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：S435.111.41

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