蜡蚧轮枝菌附着胞发育相关基因的克隆、鉴定与功能分析

Entomopathogenic fungi as a kind of insect pest pathogens have been intensively studied to develop mycoinsecticide for integration into management systems for insect pests. However, as true pathogens, all fungal biocontrol agents always kill insects more slowly than chemical insecticides. These disadvantages have often discouraged the use of fungal formulations in insect pest control. It is necessary to explore more effective means to facilitating fungal genetic improvement of virulence and knock-down speed, also accelerate the mycoinsecticide commercial production. Pathogenic infection mechanism is needed basically. Appressorium is the prerequisite and crucial for pathogens penetrate the host cuticle during the infection process. We use Lecanicillium lecanii as a model for experimental materials for genetic improvement of fungal biocontrol agents. Based on the blastspores-based genetic transformation system and Subtractive EST Library of Lecanicillium lecanii , an appressorium development required gene (LeGAS) of L. lecanii and its regulated sequences were cloned. In order to clarify the underlying mechanisms of these genes and to found new virulence genes and special promoting regulation elements during the appressorium developing process, the functions of gene LeGAS were analyzed using the techniques of RNA interfering and gene overexpression,screening for LeGAS gene expression up-regulated and down-regulated the engineering strain. Southern blot and RT-PCR technology were used to identify the expression of the LeGAS gene in the transformed strain. The physiological and biochemical characteristics and pathogenic variation of the transformed strains were detected to identify the function of LeGAS gene in the pathogenesis . Overall, the study developed a new technical platform for analysis of appressorium development required gene functions and genetic improvement of L. lecanii and other fungal biocontrol agents. The purpose of this study was trying to found new target genes, theories and techniques for genetic improvement of L. lecanii and other fungal biocontrol agents.

昆虫病原真菌杀虫剂存在杀虫速率慢的缺陷，需通过菌株遗传改良方法，来提高杀虫速率和毒力,加速其产业化。昆虫病原真菌侵染致病分子机制研究是菌株遗传改良的基础工作，侵染结构-附着胞的形成、发育是病原真菌侵染昆虫致病的前提和关键。本研究以应用广泛的蜡蚧轮枝菌为试验材料，在建立了蜡蚧轮枝菌遗传转化系统和构建了蜡蚧轮枝菌附着胞形成期间EST差减文库的基础上，克隆附着胞发育相关基因LeGAS及其调控序列；利用基因超表达技术构建LeGAS基因的超表达，利用RNAi技术转化蜡蚧轮枝菌，筛选LeGAS基因表达上调和下调的工程菌株；采用Southern杂交和RT-PCR技术，鉴定、分析转化菌株的LeGAS基因表达水平，检测转化菌株的生理生化特征及致病性的变化，鉴定LeGAS基因在致病过程中的功能。旨在发现新的毒力基因及附着孢形成期间特异启动的调控元件，为构建新型高毒力工程菌株提供新的目的基因和理论依据。

项目以应用广泛的蜡蚧轮枝菌为试验材料，在建立了蜡蚧轮枝菌遗传转化系统和构建了蜡蚧轮枝菌附着胞形成期间EST差减文库的基础上研究了以下几个方面：.（1）克隆了蜡蚧轮枝菌附着胞发育基因LeGAS全长cDNA序列；比较附着胞发育基因LeGAS与其他真菌类附着胞发育基因LeGAS序列的相似性，经克隆得到的LeGAS1和LeGAS2基因核苷酸序列，与蝗绿僵菌同源性最高为98%和99%，与稻瘟病同源性为33.3%和52.8%，小麦白粉菌同源性为31.7%和52.9%。利用3’RACE和5’RACE方法对两个基因全长cDNA进行克隆。.（2）构建附着胞发育基因LeGAS的过量表达及LeGAS基因反义质粒，并转化到蜡蚧轮枝菌中。挑选转化子，经筛选和验证，选取稳定的转化子，检测附着胞发育基因LeGAS表达上调和下调的水平。.（3）构建了LeGAS-EGFP融合表达载体，分析了LeGAS基因在蜡蚧轮枝菌不同时期的表达情况；.（4） 建立了LeGAS基因超表达和转发子及毒力分析的实验技术体系， LeGAS基因超表达和表达下调均对菌株遗传稳定性有影响。.（5）发现新的毒力基因：LeGAS基因、SOD基因家族Fe/Mn-SOD、蜡蚧轮枝菌菌株SOD基因家族Cu/Zn-SOD；测定了蜡蚧轮枝菌9株代表性菌株的疏水蛋白含量，提取分离了胞壁蛋白。.（6）克隆了MAPK 信号通路基因，分析了蜡蚧轮枝菌附着胞形成过程中相关调节基因的表达情况。.实验结果为构建新型高毒力工程菌株提供了新的目的基因和理论依据。通过菌株遗传改良，来提高杀虫速率和毒力，促进杀虫真菌的产业化。