蝗绿僵菌O-甘露糖基转移酶PMTs家族的功能分析

发布时间：2019-03-05 13:12

【摘要】：昆虫病原真菌作为重要的生防资源,阐明其侵染致病的分子机制十分必要。O-甘露糖基转移酶PMTs家族是真菌进行蛋白糖基化修饰的重要酶类,在植物病原真菌和人类病原真菌中,PMTs影响真菌的生长发育,抗逆和毒力,但在昆虫病原真菌中研究较少。本文将以模式昆虫病原真菌绿僵菌为实验材料,采用基因敲除、干扰及回复策略分析了该家族的功能。主要研究结果如下:1.Ma Pmt1/2/4的敲除和干扰根据已知的绿僵菌全基因组序列和c DNA序列,克隆得到甘露糖基转移酶PMT家族基因Ma Pmt1,Ma Pmt2,Ma Pmt4。分析该家族基因具有PMT蛋白的典型结构。构建Ma Pmt1、Ma Pmt4基因敲除载体和回复载体,转化绿僵菌,PCR筛选和Southern杂交验证获得Ma Pmt1、Ma Pmt4敲除菌株和回复菌株。构建Ma Pmt2的干扰载体,并转化绿僵菌,PCR筛选转化子,确定各干扰菌株的干扰效率。2.Ma Pmt1/2/4对绿僵菌生长发育和抗逆的影响敲除Ma Pmt1对绿僵菌的萌发和产孢量无影响;敲除Ma Pmt4后,绿僵菌萌发速度减慢,但其产孢量无变化;干扰Pmt2后,绿僵菌萌发速度减慢,产孢量仅为WT的50%左右。Ma Pmt1、Ma Pmt2、Ma Pmt4的缺失或干扰后绿僵菌对紫外和湿热的耐受能力下降。3.Ma Pmt1/2/4的缺失或干扰影响绿僵菌的毒力Ma Pmt1/2/4的缺失或干扰导致绿僵菌的毒力降低。敲除Ma Pmt1主要通过影响附着胞的膨压降低绿僵菌毒力;干扰Ma Pmt2主要通过影响附着胞的形成和膨压降低绿僵菌毒力;敲除Ma Pmt4主要通过影响附着胞的形成和膨压以及昆虫血淋巴中的生长降低绿僵菌毒力。4.Ma Pmt1/2/4的缺失或干扰,影响绿僵菌细胞壁的组分及结构通过透射电镜发现,Ma Pmt1/2/4敲除或干扰菌株细胞壁变薄且细胞壁与细胞膜分界模糊。荧光抗体染色发现,Ma Pmt1/2/4敲除或干扰菌株表面结构发生改变。细胞壁组成分析发现,Ma Pmt1/2/4缺失或干扰菌株的甘露糖蛋白、几丁质含量显著低于野生型菌株,而葡聚糖含量与野生型相比无显著差异。该研究结果对于明确O-甘露糖基化修饰在绿僵菌中的作用,特别是O-甘露糖基化修饰的改变对绿僵菌细胞壁重要成分、结构及侵染致病能力的影响,以及细胞壁组成、结构与昆虫病原真菌侵染致病的关系具有重要意义。
[Abstract]:As an important biocontrol resource, it is necessary to clarify the molecular mechanism of entomopathogenic fungi infection. O-mannose transferase PMTs family is an important enzyme for protein glycosylation modification of fungi. In plant pathogenic fungi and human pathogenic fungi, PMTs affects the growth and development, stress resistance and virulence of fungi, but few studies have been done in entomopathogenic fungi. Using the model entomopathogenic fungus Metarhizium anisopliae as the experimental material, the function of the family was analyzed by gene knockout, interference and recovery strategy. The main results are as follows: according to the known whole genome sequence and c DNA sequence of Metarhizium anisopliae, the gene Ma Pmt1,Ma Pmt2,Ma Pmt4. of mannose transferase PMT family was cloned by knockout and interference of 1.Ma Pmt1/2/4 in Metarhizium anisopliae. The typical structure of PMT protein was analyzed. Construction of Ma Pmt1,Ma Pmt4 gene knockout vector and recovery vector, transformation of Metarhizium anisopliae, PCR screening and Southern hybridization to obtain Ma Pmt1,Ma Pmt4 knockout strain and recovery strain. The interference vector of Ma Pmt2 was constructed and transformed into Metarhizium anisopliae. The transformants were screened by PCR. 2. The effect of Ma Pmt1/2/4 on growth, development and stress resistance of Metarhizium anisopliae. Knockout of Ma Pmt1 had no effect on germination and sporulation of Metarhizium anisopliae. After knockout of Ma Pmt4, the germination rate of Metarhizium anisopliae decreased, but the sporulation of Metarhizium anisopliae remained unchanged. After interfering with Pmt2, the germination rate of Metarhizium anisopliae slowed down, and the spore yield was only about 50% of that of WT. Ma Pmt1,Ma Pmt2, The resistance of Metarhizium anisopliae to ultraviolet and wet heat decreased after the deletion or interference of Ma Pmt4. The deletion or interference of 3.Ma Pmt1/2/4 affected the virulence of Metarhizium anisopliae. The deletion or interference of Ma Pmt1/2/4 resulted in the decrease of virulence of Metarhizium anisopliae. Knockout of Ma Pmt1 decreased the virulence of Metarhizium anisopliae mainly by affecting the inflating pressure of appressorium and interfering with Ma Pmt2 by affecting the formation of appressorium and expanding pressure to decrease the virulence of Metarhizium anisopliae. Knockout of Ma Pmt4 decreased the virulence of Metarhizium anisopliae mainly by affecting appressorium formation and swelling and the growth of insect hemolymph. 4. The deletion or interference of Ma Pmt1/2/4 and the composition and structure of cell wall of Metarhizium anisopliae were found by transmission electron microscopy. The cell wall of Ma Pmt1/2/4 knockout or interference strain became thinner and the boundary between cell wall and cell membrane was blurred. Fluorescent antibody staining showed that the surface structure of Ma Pmt1/2/4 knockout or interfering strain was changed. Cell wall composition analysis showed that the mannose protein and chitin content of Ma Pmt1/2/4-deficient or interfering strains were significantly lower than that of wild-type strains, but there was no significant difference in glucan content between wild-type strains and wild-type strains. The results of this study can be used to clarify the role of O-mannose glycosylation in Metarhizium anisopliae, especially the effect of O-mannose modification on the important components, structure and invading ability of Metarhizium anisopliae cell wall, as well as the composition of cell wall. The relationship between the structure of entomopathogenic fungi and the infection of entomopathogenic fungi is of great significance.
【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】：S476.1

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