构建香菇转化的同源筛选标记的研究  

Construction of Homologous Carboxin Resistant Selection Marker in Lentinula edodes

作　　者：李菲 章炉军 姜宁 刘建雨 于海龙 尚晓冬 宋春艳 谭琦 LI Fei;ZHANG Lujun;JIANG Ning;LIU Jianyu;YU Hailong;SHANG Xiaodong;SONG Chunyan;TAN Qi(College of Food Science&Technology,Shanghai Ocean University,Shanghai 201306,China;Institute of Edible Fungi,Shanghai Academy of Agricultural Sciences,Key Laboratory of Edible Fungi Resourcesand Utilization(South)Ministry of Agriculture,P.R China,National Engineering Research Center of Edible Fungi,National R&D Center for Edible Fungi Procesing,Key Laboratory of Agricultural Genetics and Breeding of Shanghai,Shanghai 201403,China)

机构地区：[1]上海海洋大学食品学院 [2]上海市农业科学院食用菌研究所农业部南方食用菌资源利用重点实验室国家食用菌工程技术研究中心国家食用菌加工技术研发分中心上海市农业遗传育种重点开放实验室

出　　处：《食用菌学报》2019年第4期16-22,共7页Acta Edulis Fungi

基　　金：国家现代农业产业技术体系建设专项(CARS-20);上海市食用菌产业技术体系建设专项[沪农科产字(2018)第9-03号)];上海市科委项目(18391900200)

摘　　要：通过比对分析多种导致真菌内源萎锈灵抗性的琥珀酸脱氢酶B亚基(SdhB)氨基酸序列的突变位点,将香菇(Lentinula edodes)SdhB氨基酸序列第243位的组氨酸替换为亮氨酸,构建了香菇萎锈灵抗性同源筛选标记载体,以农杆菌介导转化小米粒培养的香菇菌丝。转化子经5次传代后,PCR扩增验证插入片段,并观察绿色荧光蛋白(EGFP)的表达情况。结果显示:香菇单核体和双核体菌株经转化后,均能筛选到具有萎锈灵抗性的转化子,单核体菌株的转化率为34%,显著高于双核体菌株(4%);单核体转化子中的同源筛选标记及报告基因稳定性较好。本研究构建的萎锈灵抗性同源筛选标记可有效地在香菇遗传转化体系中应用。A plasmid bearing a homologous carboxin resistant selection maker Cbx^R and the gene of enhanced green fluorescent protein(EGFP)was constructed and transferred to mycelia of Lentinula edodes cultivated on millet using Agrobacterium-mediated transformation.This homolougous carboxin resistant selection maker Cbx^R was generated by replacing the histidine at the 243 rd amino acid of the succinic dehydrogenase B subunit(SdhB)to leucine.Positive transformants were subcultured for 5 passages and then verified by PCR and EGFP expression.The results showed that both monokaryon and dikaryon were successfully integrated with Cbx^R with transformation efficiencies of 34%and 4%,respectively.The transformation efficiency of monokaryon was significantly higher than that of dikaryon.The insertion of Cbx^R and EGFP expression were stable in monokaryon transformants.This homologous carboxin resistant marker Cbx^R can be used effectively in transformation of L.edodes.

关 键 词：香菇 萎锈灵抗性基因 同源筛选标记 农杆菌转化 绿色荧光蛋白 

分 类 号：S64[农业科学—蔬菜学]