绿僵菌桔霉素生物合成氧化还原酶基因MaCtnB的克隆与功能分析
发布时间：2019-02-15 06:54
【摘要】：绿僵菌(Metarhizium anisopliae)作为一类重要昆虫病原真菌生防潜力巨大。在自然条件下,绿僵菌主要以孢子控制害虫。孢子成熟时间的长,生产成本高,易受环境的影响,杀虫速度慢等都制约真菌农药的大规模生产及其应用。在高温、营养缺陷等不利条件下,自身能进行微循环产孢。微循环产孢是丝状真菌遇到逆境条件的一种自我保护机制,具有产孢速度快,产孢量大,孢子大小更为均一等优点,但目前该机制的相关基因研究报道尚少。为了明确微循环产孢的机制,我们课题组在前期构建了绿僵菌微循环产孢时期的均一化全长c DNA文库和微循环产孢时期的差减文库,发现桔霉素生物合成氧化还原酶Ctn B基因在上述文库中上调表达。根据绿僵菌全基因组序列克隆得到桔霉素生物合成氧化还原酶基因Ma Ctn B,并采用基因敲除策略对该基因功能进行研究,主要研究结果如下:①Ma Ctn B影响绿僵菌在微循环产孢培养基上的的产孢方式和产孢量,以及影响了绿僵菌在正常培养基上的萌发;②桔霉素影响了绿僵菌的产孢方式;③Ma Ctn B影响绿僵菌对紫外辐射和湿热的敏感性,但不影响细胞壁完整性、抗氧化还原能力以及离子渗透压的耐受性;④Ma Ctn B影响绿僵菌的毒力:其机制是绿僵菌在寄主体壁穿透阶段中的萌发、附着胞的形成、膨压有一定影响;但主要在体内定殖阶段,通过影响绿僵菌在寄主体内虫菌体的生长,寄主血细胞的数量以及血细胞的吞噬作用来影响寄主的先天免疫,从而降低了毒力。
[Abstract]:Metarhizium anisopliae (Metarhizium anisopliae) has great biocontrol potential as an important class of entomopathogenic fungi. Under natural conditions, Metarhizium anisopliae mainly uses spores to control pests. The long maturation time of spores, high production cost, easy to be affected by environment and slow insecticidal rate all restrict the large-scale production and application of fungal pesticides. Under such unfavorable conditions as high temperature and nutritional deficiency, microcirculation sporulation can be carried out. Microcirculatory sporulation is a self-protective mechanism of filamentous fungi under adverse conditions. It has the advantages of fast sporulation, large sporulation and more uniform spores size. However, there are few studies on the genes related to this mechanism. In order to clarify the mechanism of microcirculation sporulation, our group constructed a homogeneous full-length c DNA library and a differential subtractive library during microcirculatory sporulation stage of Metarhizium anisopliae. It was found that Ctn B gene of citrinin biosynthesis redox enzyme was up-regulated in the above library. According to the whole genome sequence of Metarhizium anisopliae, the Ma Ctn B, gene of citrinin biosynthesis redox enzyme was cloned and the function of the gene was studied by gene knockout strategy. The main results were as follows: 1Ma Ctn B affected the sporulation mode and sporulation quantity of Metarhizium anisopliae on microcirculation sporulation medium and the germination of Metarhizium anisopliae on normal medium. (2) citrinin affected the sporulation of Metarhizium anisopliae, 3Ma Ctn B affected the sensitivity of Metarhizium anisopliae to ultraviolet radiation and damp heat, but it did not affect the integrity of cell wall, the ability of antioxidant reduction and the tolerance of ion osmotic pressure. 4Ma Ctn B affects the virulence of Metarhizium anisopliae: the mechanism is the germination of Metarhizium anisopliae in the stage of wall penetration, the formation of appura, and the influence of swelling pressure. But in the colonization stage, the innate immunity of the host was affected by affecting the growth of Metarhizium anisopliae in the host, the number of the host blood cells and the phagocytosis of the blood cells, thus reducing the virulence of the host.
【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
本文编号：2423079
[Abstract]:Metarhizium anisopliae (Metarhizium anisopliae) has great biocontrol potential as an important class of entomopathogenic fungi. Under natural conditions, Metarhizium anisopliae mainly uses spores to control pests. The long maturation time of spores, high production cost, easy to be affected by environment and slow insecticidal rate all restrict the large-scale production and application of fungal pesticides. Under such unfavorable conditions as high temperature and nutritional deficiency, microcirculation sporulation can be carried out. Microcirculatory sporulation is a self-protective mechanism of filamentous fungi under adverse conditions. It has the advantages of fast sporulation, large sporulation and more uniform spores size. However, there are few studies on the genes related to this mechanism. In order to clarify the mechanism of microcirculation sporulation, our group constructed a homogeneous full-length c DNA library and a differential subtractive library during microcirculatory sporulation stage of Metarhizium anisopliae. It was found that Ctn B gene of citrinin biosynthesis redox enzyme was up-regulated in the above library. According to the whole genome sequence of Metarhizium anisopliae, the Ma Ctn B, gene of citrinin biosynthesis redox enzyme was cloned and the function of the gene was studied by gene knockout strategy. The main results were as follows: 1Ma Ctn B affected the sporulation mode and sporulation quantity of Metarhizium anisopliae on microcirculation sporulation medium and the germination of Metarhizium anisopliae on normal medium. (2) citrinin affected the sporulation of Metarhizium anisopliae, 3Ma Ctn B affected the sensitivity of Metarhizium anisopliae to ultraviolet radiation and damp heat, but it did not affect the integrity of cell wall, the ability of antioxidant reduction and the tolerance of ion osmotic pressure. 4Ma Ctn B affects the virulence of Metarhizium anisopliae: the mechanism is the germination of Metarhizium anisopliae in the stage of wall penetration, the formation of appura, and the influence of swelling pressure. But in the colonization stage, the innate immunity of the host was affected by affecting the growth of Metarhizium anisopliae in the host, the number of the host blood cells and the phagocytosis of the blood cells, thus reducing the virulence of the host.
【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
【参考文献】
相关期刊论文 前1条
1 马俊义;李尽哲;叶兆伟;;生物农药的应用现状及前景展望[J];江苏农业科学;2011年04期
，本文编号：2423079
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