摘要
丝状真菌分泌蛋白与其致病性密切相关,目前对于病原真菌的蛋白胞外分泌途径及其调控机制的报道不多。为建立一个方便高效的真菌分泌蛋白调控途径的遗传研究体系,本研究以植物病原丝状真菌——板栗疫病菌寄生隐赤壳Cryphonectria parasitica为对象,选取分泌表达量最高的两个分泌蛋白的信号肽SP1和SP2,分别构建带有GUS报告基因的分泌蛋白表达载体p CPXBle-SP1-GUS和p CPXBle-SP2-GUS并用于转化板栗疫病菌。选择高效分泌GUS蛋白的转化株SP1-9为出发菌株,利用农杆菌介导的遗传转化技术构建了T-DNA插入突变体库,从576个突变体中筛选到2株GUS分泌表达明显降低的突变体。本研究成功构建了可用于研究丝状真菌蛋白分泌的遗传研究体系,并筛选获得了分泌蛋白缺陷突变体,为深入研究丝状真菌分泌途径及其调控机制奠定了基础。
Secreted proteins of filamentous fungi are closely related to fungal pathogenicity.At present,there are few reports on the protein secretion pathway and the regulatory mechanism(s)in pathogenic fungi.For establishing a convenient and efficient genetic research system for investigating the regulation of fungal secretory proteins in Cryphonectria parasitica that causes chestnut blight,the reporter vectors for secretory expression were constructed based on our previous secretory proteomics analysis by using two signal peptides SP1 and SP2 with the highest expression level,to fuse with the GUS gene.The resulting constructs,namely pCPXBle-SP1-GUS and pCPXBle-SP2-GUS,were then transformed into the wild-type C.parasitica strain EP155.The transformant strain SP1-9 showing the highest secreted GUS efficiency was used to generate the T-DNA insertion mutants by Agrobacterium tumefaciens-mediated transformation(ATMT)method.Out of the 576 such T-DNA insertion mutants,two mutants significantly decreased GUS secretion.This study successfully established a high-throughput transformation system for investigating the secretory pathway in filamentous fungi,and obtained mutants having defect of protein secretion,being beneficial to future studies on molecular mechanism of fungal secretory pathway.
作者
李茹
仇金凤
杨彦彦
王金子
陈凤月
陈保善
LI Ru;QIU Jin-Feng;YANG Yan-Yan;WANG Jin-Zi;CHEN Feng-Yue;CHEN Bao-Shan(State Key Laboratory for Conservation and Utilization of Subtropical Agro-bioresources,College of Life Science and Technology,Guangxi University,Nanning,Guangxi 530004,China;College of Agriculture,Guangxi University,Nanning,Guangxi 530004,China;School of Ocean and Biotechnology,Guangxi University for Nationalities,Nanning,Guangxi 530006,China)
出处
《菌物学报》
CAS
CSCD
北大核心
2021年第7期1688-1699,共12页
Mycosystema
基金
国家自然科学基金(31760498)。
关键词
寄生隐赤壳
分泌途径
GUS报告基因
根癌农杆菌
Cryphonectria parasitica
secretory pathway
reporter gene GUS
Agrobacterium tumefaciens
