越南假拟盘多毛孢转化体系构建  

Construction of a transformation system for Pseudopestalotiopsis vietnamensis

作　　者：刘午鸿 平均雄 齐赛洋 罗心阳 郭依娜 戴方妍 傅仙玉 武广 LIU Wuhong;PING Junxiong;QI Saiyang;LUO Xinyang;GUO Yina;DAI Fangyan;FU Xianyu;WU Guangheng(College of Ecology and Resources Engineering,Wuyi University,Wuyishan 354300,Fujian,China;Fujian Provincial Key Laboratory of Eco-Industrial Green Technology,Wuyi University,Wuyishan 354300,Fujian,China;College of Tea and Food Science,Wuyi University,Wuyishan 354300,Fujian,China)

机构地区：[1]武夷学院生态与资源工程学院,福建武夷山354300 [2]武夷学院福建省生态产业绿色技术重点实验室,福建武夷山354300 [3]武夷学院茶与食品学院,福建武夷山354300

出　　处：《菌物学报》2025年第4期59-67,共9页Mycosystema

基　　金：南平市资源化学产业科技创新联合资助项目(N2020Z007);福建省自然科学基金(2022J011200)。

摘　　要：越南假拟盘多毛孢引致茶轮斑病,该病在武夷山茶树多发,严重影响武夷山茶叶品质。为能够深入研究越南假拟盘多毛孢致病机理,我们对其原生质体转化体系开展研究。以分离纯化的越南假拟盘多毛孢为对象,利用酶解法分离得到原生质体,采用pSULPH质粒作为载体,借助PEG-Ca^(2+)介导的方法,将目标质粒转入原生质体中促使其再生,最后通过共聚焦激光扫描显微镜及蛋白质提取对带有GFP的转化菌株进行验证。结果发现,该病原菌使用CM培养基时菌丝抱团严重,而将培养基由CM更换为PD后菌丝抱团得到缓解,提升了酶解效率,为获取高质量的原生质体提供了有力支持;利用PEG-Ca^(2+)介导的转化方法,成功地将绿色荧光蛋白基因转入越南假拟盘多毛孢中,得到荧光强度均一且遗传稳定的转化子。综上所述,该研究构建了越南假拟盘多毛孢原生质体转化体系,为进一步研究其致病基因功能奠定了基础。Pseudopestalotiopsis vietnamensis is widespread in the tea gardens in Wuyi Mountain,causing gray blight disease(GBD)that severely affects the quality of tea.For understanding the pathogenic mechanism of Ps.vietnamensis,the research on protoplast transformation system of the fungus was conducted.Protoplasts of purified Ps.vietnamensis were isolated by using an enzymatic digestion method.pSULPH plasmid was employed as a vector,and the target plasmid was introduced into the protoplasts via PEG-Ca^(2+)-mediated transformation to promote its regeneration.Finally,the GFP-transformed strains were validated through confocal laser scanning microscopy and protein extraction.It was found that the mycelia of Ps.vietnamensis exhibited severe clumping on CM medium.The clumping appearance could be alleviated by switching the mycelia to PD medium,and thereby improving the efficiency of enzymatic digestion and providing strong support for obtaining high-quality protoplasts.Using the PEG-Ca^(2+)-mediated transformation method,the green fluorescent protein gene was successfully introduced into Ps.vietnamensis strain CLBB1,resulting in the acquirement of transformant with uniform fluorescence intensity and genetic stability.This study lays the foundation for further research into the functions of Ps.vietnamensis pathogenic genes.

关 键 词：越南假拟盘多毛孢 原生质体 PEG-Ca^(2+)介导 GFP转化 茶轮斑病 

分 类 号：S435.711[农业科学—农业昆虫与害虫防治]