蝗绿僵菌GTPase激活蛋白基因Gyp3的功能研究

发布时间：2018-06-11 13:40

本文选题：绿僵菌 + GTP酶活化蛋白　；参考：《重庆大学》2015年硕士论文

【摘要】：绿僵菌是一类重要的昆虫病原真菌,具有生防菌株的共有优点;同时由于其宿主广泛而成为研究昆虫病原真菌的模式生物。绿僵菌在日常生产生活施用中也存在诸多制约因素,比如受环境影响大、杀虫速度较慢等。因此对影响昆虫病原真菌毒力和抗逆境能力的基因进行研究并以此为突破口改良菌株,能为真菌农药的开发利用提供重要的理论基础。研究表明GYP3通过抑制Rab5家族蛋白活性从而降低GTP水解速率,在病原真菌中并没有对该蛋白的研究报道。同时在病原真菌中研究发现该类蛋白的靶蛋白Rab5家族对致病真菌的正常生长,毒力和抗逆境能力都至关重要;有报道称Rab5家族在压力条件下发挥作用时需要Rab5 GAP蛋白的参与,那么GYP3或许可以通过调控Rab5家族活性从而实现对菌株毒力和抗逆境能力等方面的影响,所以本研究为发现Gyp3基因在昆虫病原真菌中的功能及作用机制提供了理论证据,同时这也有利于利用分子生物学与基因工程等手段实现对绿僵菌菌株的改良。本文以绿僵菌作为实验材料,通过对该基因敲除及回复明确该基因功能分析其作用机制。主要研究结果如下:1、Gyp3降低绿僵菌对Na+和对湿热以及紫外辐射(UV-B)的耐受能力:在加入Nacl的1/4 SDA固体培养基上,敲除转化子菌落大小明显小于野生型和回复转化子;随着UV-B和热激时间的增加,ΔGyp3与WT和CP相比萌发率显著降低。2、Gyp3不调控绿僵菌产孢量,孢子附着胞形成率及对细胞壁破坏剂耐受性:在加入细胞壁破坏剂的1/4 SDA固体培养基上,敲除转化子菌落大小与野生型和回复转化子相比无明显差异。通过测量统计,ΔGyp3的产孢量和孢子附着胞形成率与WT和CP相比降低无显著差异。3、Gyp3通过影响绿僵菌在东亚飞蝗体表的萌发率、膨压及附着胞水解体壁水解酶的表达、在寄主昆虫血淋巴中的生长等过程调控其毒力:通过测量统计,ΔGyp3的萌发率、膨压及附着胞水解体壁水解酶的表达与WT和CP相比显著降低;抑制在寄主昆虫血淋巴中的生长。4、Gyp3可以通过调控靶蛋白家族Rab家族的表达,进而实现对Rab蛋白活性的抑制:通过测量统计,ΔGyp3的靶蛋白家族Rab家族的表达与WT和CP相比显著降低。
[Abstract]:Bacillus anisopliae is an important class of entomopathogenic fungi. It has the common advantages of biocontrol strains. At the same time, because of its extensive host, it has become a model organism for the study of insect pathogenic fungi. The gene of fungal virulence and resistance to adversity has been studied and used as a breakthrough strain to provide an important theoretical basis for the development and utilization of fungal pesticides. The study shows that GYP3 can reduce the rate of GTP hydrolysis by inhibiting the activity of Rab5 family protein, and there is no report on the protein in the pathogenic fungi. The study found that the target protein Rab5 family of this protein is essential for the normal growth of pathogenic fungi, virulence and resistance to adversity; it is reported that the Rab5 family needs the participation of Rab5 GAP protein in stress conditions, and then GYP3 may be able to regulate the virulence and adversity of the strain by regulating the activity of the Rab5 family. This study provides theoretical evidence for the discovery of the function and mechanism of the Gyp3 gene in the entomopathogenic fungi, and it is also beneficial to the improvement of Bacillus anisopliae by means of molecular biology and genetic engineering. The main research results are as follows: 1, Gyp3 reduces the tolerance of Bacillus anisopliae to Na+ and humid heat and ultraviolet radiation (UV-B): on the 1/4 SDA solid medium with Nacl, the size of the knockout transformant colony is obviously smaller than the wild type and the return transformant; with the increase of UV-B and heat shock time, Delta Gyp3 Compared with WT and CP, the germination rate significantly decreased.2, Gyp3 did not regulate the sporulation of Bacillus anisopliae, the formation rate of spores and the tolerance to the cell wall destruction agent: there was no significant difference in the ratio of the colonies of the transformants to the wild type and the reverse transformant on the 1/4 SDA solid medium added to the cell wall destructive agent. By measurement, the sporulation of delta Gyp3 There was no significant difference in the formation rate of the spores and spore attachments compared with the WT and CP,.3. Gyp3 regulates the virulence by influencing the germination rate of the body surface of the oriental migratory locust, the expression of the swelling pressure and the hydrolase of the disintegrated wall of the attached cell water, and the growth of the host insect's haemolymph: the germination rate of delta Gyp3, the swelling pressure and the attached cell water through the measurement and statistics. The expression of the disintegrated wall hydrolase was significantly lower than that of WT and CP, and the inhibition of the growth of.4 in the haemolymph of host insects, Gyp3 could inhibit the activity of Rab protein by regulating the expression of the Rab family in the target protein family, and the expression of the Rab family of the target protein family of the delta Gyp3 was significantly lower than that of WT and CP.
【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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