蝗绿僵菌几丁质合成酶家族的功能研究

发布时间：2018-03-22 12:48

本文选题：蝗绿僵菌　切入点：几丁质合成酶家族　出处：《重庆大学》2015年硕士论文　论文类型：学位论文

【摘要】：随着现代分子生物学技术的不断发展,有关昆虫病原真菌致病机理的研究越来越深入。蝗绿僵菌是目前研究比较广的一类昆虫病原真菌,可以作为研究昆虫病原真菌的模式生物。几丁质是真菌细胞壁的重要组成部分,几丁质合成的关键酶是几丁质合酶(CHS)。在丝状真菌中,几丁质合成酶家族基因分为七类。目前,有关昆虫病原真菌几丁质合成酶家族基因的研究还未见报道。所以本研究以昆虫病原真菌蝗绿僵菌为研究对象,研究蝗绿僵菌几丁质合成酶家族基因Ma Chs,阐明Ma Chs家族各基因在蝗绿僵菌抗逆和侵染致病过程中的作用。本研究的主要研究结果如下:①蝗绿僵菌Ma Chs家族有七个基因,分别命名为Ma Chs I、Ma Chs II、Ma Chs III、Ma Chs IV、Ma Chs V、Ma Chs VI、Ma Chs VII。②通过q RT-PCR测定Ma Chs家族各基因在侵染过程中的表达量。除5天虫菌体中Ma Chs VII表达量最高,其余各时期均是Ma Chs III相对表达量最高,Ma Chs VI在各时期相对表达量最低;比较同一基因在不同时期的表达量,Ma Chs II在附着胞时期相对表达量最高,其他各基因均在孢子时期相对表达量最高。③在1/4SDAY固体培养基加入各种逆境,ΔMa Chs III、ΔMa Chs V在CR、CFW和H2O2条件下,长势弱于野生型,ΔMa Chs VII在CR、CFW、H2O2、SDS、SOR、Na Cl逆境及1/4SDAY空白对照中长势均弱于野生型,其他敲除菌株在逆境下与野生型没有差异。敲除Ma Chs II、Ma Chs III、Ma Chs IV、Ma Chs V和Ma Chs VI影响蝗绿僵菌对湿热的敏感性,敲除Ma Chs II、Ma Chs IV、Ma Chs VI影响蝗绿僵菌对紫外的敏感性,ΔMa Chs I对紫外湿热的敏感性与野生型无差异。④分析Ma Chs家族各基因对蝗绿僵菌产孢量和萌发的影响。结果显示,除ΔMa Chs I外,其他敲除菌株产孢量均比野生型低,其中ΔMa Chs VII产孢量急剧降低。统计萌发率显示ΔMa Chs I、ΔMa Chs IV萌发中时延迟;ΔMa Chs II、ΔMa Chs III、ΔMa Chs V萌发中时提前,ΔMa Chs VI萌发中时与野生型相比没有显著差异。⑤通过点滴和注射分析各基因敲除后对蝗绿僵菌毒力的影响。结果显示ΔMa Chs III点滴和注射接种蝗虫,毒力均降低。研究发现,ΔMa Chs V注射毒力升高,ΔMa Chs VII注射毒力丧失,其余各基因对蝗绿僵菌毒力没有影响。⑥通过透射电镜观察Ma Chs家族各基因敲除后对蝗绿僵菌细胞壁的影响,结果显示敲除菌株细胞壁与野生型有一定的差异,敲除菌株细胞壁变厚。
[Abstract]:With the development of modern molecular biology technology, the pathogenic mechanism of entomopathogenic fungi has been studied more and more deeply. Metarhizium anisopliae is a kind of insect pathogenic fungi. It can be used as a model organism to study entomopathogenic fungi. Chitin is an important part of the cell wall of fungi, and the key enzyme for chitin synthesis is chitin synthase (CHS). In filamentous fungi, the genes of chitin synthase family are classified into seven categories. Studies on chitinase family genes of entomopathogenic fungi have not been reported. To study the genes of chitin synthase family of Metarhizium anisopliae Ma, and to elucidate the role of Ma Chs family genes in the process of resistance to stress and infection and pathogenicity of Metarhizium anisopliae. The main results of this study are as follows: 1. There are seven genes in the Ma Chs family of Metarhizium anisopliae. They were named as Ma Chs, Chs, Chs, Chs, Chs, Chs, Chs, Chs, Chs, Chs, Chs, Chs VII.2, respectively. The expression of Ma Chs VII in the Ma Chs family was determined by Q RT-PCR, except for 5 days, the highest expression was found in Ma Chs VII. The highest relative expression of Ma Chs III and the lowest relative expression of Ma Chs VI were observed in other periods, and the highest relative expression of Ma Chs II was observed at different stages. The relative expression of other genes was highest in spore stage. 3. 3. All the stresses were added to 1/4SDAY solid medium. 螖 Ma Chs III, 螖 Ma Chs V were weaker than wild type in CRF CFW and H2O2, and 螖 Ma Chs VII were weaker than wild type in CRT CFW H 2O 2 SDSS-SORN NaCl stress and 1/4SDAY blank control. Other knockout strains had no difference from wild type under stress. Knockout of Ma Chs III Chs Ma Chs IVP Ma Chs V and Ma Chs VI affected susceptibility of Metarhizium anisopliae to moisture and heat. Knockout Ma Chs II, Ma Chs IVF Ma Chs VI affected the UV sensitivity of Metarhizium anisopliae. There was no difference between the sensitivity of 螖 Ma Chs I to ultraviolet dampness heat and wild type. 4. The effect of Ma Chs family genes on sporulation and germination of Metarhizium anisopliae was analyzed. The results showed that, except for 螖 Ma Chs I, there was no difference between the two genes. The sporulation of other knockout strains was lower than that of wild type. The spore production of 螖 Ma Chs VII decreased sharply. The statistical germination rate showed that the germination time of 螖 Ma Chs I, 螖 Ma Chs IV was delayed, that of 螖 Ma Chs II, 螖 Ma Chs III, 螖 Ma Chs V was earlier, and there was no significant difference between 螖 Ma Chs VI and wild type. The effect of gene knockout on the virulence of Metarhizium anisopliae was analyzed. The results showed that 螖 Ma Chs III was given by drip and inoculated with locust. The virulence of 螖 Ma Chs V was increased, and the toxicity of 螖 Ma Chs VII was lost. The other genes had no effect on the virulence of Metarhizium anisopliae by transmission electron microscope (TEM). The effect on the cell wall of Metarhizium anisopliae was observed after the gene knockout of Ma Chs family. The results showed that the cell wall of the knockout strain was different from that of the wild type, and the cell wall of the knockout strain became thicker.
【学位授予单位】：重庆大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S476.12

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