板栗疫病菌核糖体蛋白相关基因RPS18、RPL30、RPL32-B的克隆及其功能研究.docVIP

 n 板栗疫病菌核糖体蛋白相关基因 RPS18、 RPL30、RPL32-B 的克隆及其功能研究# 李峰1，林海燕1，朱超1，施李鸣1，商巾杰1,2,3，陈保善1,2,3** 5 10 15 20 25 30 35 40 （1. 广西大学生命科学与技术学院，南宁 530005； 2. 亚热带农业生物资源保护与利用国家重点实验室，南宁 530005； 3. 微生物及植物遗传工程教育部重点实验室，南宁 530005） 摘要：低毒病毒/板栗疫病菌系统是研究病毒与宿主相互作用分子机制以及植物病原真菌致 病机制的一个模式系统。本研究尝试对受低毒病毒调控的核糖体蛋白 RPS18、RPL30 和 RPL32-B 三个基因分别进行基因敲除，但仅 RPL32-B 得到为数不多的转化子，且只能以杂合 子的形式存在，表明这些核糖体基因在板栗疫病菌中可能是必需的。构建了多核糖体基因 RNA 干扰质粒 pFCNH-intron-sll 并导入板栗疫病菌。核糖体 RNA 干扰导致宿主真菌致病力 明显降低，表明 RPS18、RPL30 和 RPL32-B 或其中之一参与板栗疫病菌致病力的调节。 关键词：板栗疫病菌；核糖体蛋白；基因敲除；多基因 RNA 干扰；致病力 中图分类号：S436.64 The Cloning and Functional Characterization of the Ribosomal Protein-Related Genes RPS18, RPL30 , and RPL32-B in Cryphonectria parasitica LI Feng1, LIN Haiyan1, ZHU Chao1, SHI Liming1, SHANG Jinjie1,2,3, CHEN Baoshan1,2,3 (1. College of Life Science and Technology,Guangxi University, NanNing 530005; 2. State Key Laboratory for Conservation and Utilization of Subtropical Agro-bioresources, NanNing 530005; 3. The Key Laboratory of Ministry of Education of China for Microbial and Plant Genetic Engineering, NanNing 530005) Abstract: Hypovirus/Cryphonectria parasitica system is a model system for investigation of virus-host interaction and mechanism of pathogenicity in fungi. In this paper, we tried to knockout three ribosomal protein (RP)-related genes RPS18, RPL30, and RPL32-B, whose expression are regulated by hypovirus infection. However, only knockout mutants for RPL32-B were obtained. These mutants were heterozygous that could not be further purified to homozygous, demonstrating that these three RP genes are indespensible in C. parasitica. An RNAi silencing recombinant plasmid pFCNH-intron-sll targetting RPS18, RPL30, and RPL32-B was developed and introduced into C. parasitica. Transformants showed significant reduction in virulence to chestnut stems, demonstrating that one or all of RPS18, RPL30, and RPL32-B is involved in virulence regulation in C. parasitica. Keywords: Cryphonectria parasitica; Ribosomal protein; Gene knockout