农杆菌介导鸡[土从]菌遗传转化体系构建  

Construction of Agrobacterium-Mediated Transformation System in Termitomyces sp.J132

作　　者：张明星 尹靖欣 李丹妮 许卓茗 徐学锋[1] ZHANG Mingxing;YIN Jingxin;LI Danni;XU Zhuoming;XU Xuefeng(College of Food Science and Technology,South China Agricultural University,Guangzhou 510642,Guangdong,China)

机构地区：[1]华南农业大学食品学院,广东广州510642

出　　处：《食用菌学报》2021年第5期12-19,共8页Acta Edulis Fungi

基　　金：广东省自然科学基金(2018A030313011);广东省重点领域研发计划(2018B020206001)。

摘　　要：采用含有双元质粒Plasmid4的3个农杆菌菌株(EHA105、GV3101和LBA4404)介导转化鸡[土从]菌(Termitomyces sp.)菌丝体,对转化体系相关的抗生素质量浓度、受体材料(菌丝球和菌丝块)、农杆菌菌株、农杆菌菌液浓度、侵染时间、共培养时间、共培养温度进行优化;并通过PCR鉴定转化子、荧光蛋白表达验证转化子的遗传稳定性。结果表明:潮霉素(hygromycin,Hyg)最适质量浓度为50μg·mL^(-1);菌丝球的转化效率优于菌丝块;EHA105菌液D600为0.7、侵染时间30 min、共培养时间48 h、共培养温度26℃时,鸡[土从]菌遗传转化效率最高。增强型荧光蛋白(enhanced green fluorescent protein,eGFP)的表达结果说明外源GFP基因已经成功整合到鸡[土从]菌基因组并获得高效稳定表达。构建的鸡[土从]菌高效转化体系为其后续相关功能基因研究和共生机制解析提供参考。Using the binary plasmid Plasmid4 and three strains of Agrobacterium tumefaciens,LBA4404,GV3101 and EHA105,a genetic transformation system was constructed in Termitomyces sp.J132.Both hyphal blocks and hyphal pellets were tested as the receptor materials,and the transformation conditions including antibiotic concentration,A.tumefaciens strain and concentration,infection time,co-culture time and co-culture temperature were optimized.The resultant transformants were verified by PCR,and their genetic stability were examined by observation of eGFP expression.The results showed that the transformation efficiency of hyphal pellets was higher than that of hyphal blocks,and the lowest lethal concentration of hygromycin was 50μg·mL^(-1).The highest transformation rate was obtained under the following conditions:A.tumefaciens EHA105 harboring Plasmid4 at D6000.7,infection for 30 min,and co-culture at 26℃for 48 h.Observation of eGFP showed that the exogenous GFP gene was successfully integrated into the genome of Termitomyces sp.J132 and expressed stably.This transformation system provided a basis for subsequent research on development-related functional genes and symbiotic mechanism of Termitomyces spp.

关 键 词：鸡[土从]菌 遗传转化 农杆菌 潮霉素 共培养 

分 类 号：TS219[轻工技术与工程—粮食、油脂及植物蛋白工程]