香蕉果实酵母双杂交文库的构建及后熟转录因子MaMADS2互作蛋白筛选  

Construction of yeast two-hybrid library and screening of interacting proteins of ripening regulator MaMADS2 in banana fruit

作　　者：王川 盛鸥[1] 窦同心[1] 李斌[1] 胡春华[1] 杨乔松[1] 邓贵明[1] 董涛[1] 李春雨[1] 易干军[1] 毕方铖[1] WANG Chuan;SHENG Ou;DOU Tongxin;LI Bin;HU Chunhua;YANG Qiaosong;DENG Guiming;DONG Tao;LI Chunyu;YI Ganjun;BI Fangcheng(Institute of Fruit Tree Research/Key Laboratory of South Subtropical Fruit Tree Biology and Genetic Resources Utilization,Ministry of Agriculture and Rural Affairs/Guangdong Key Laboratory of Tropical and Subtropical Fruit Research,Guangdong Academy of Agricultural Sciences,Guangzhou 510640,China;College of Horticulture and Forestry,Huazhong Agricultural University,Wuhan 430070,China)

机构地区：[1]广东省农业科学院果树研究所/农业农村部南亚热带果树生物学与遗传资源利用重点实验室/广东省热带亚热带果树研究重点实验室,广东广州510640 [2]华中农业大学园艺林学院,湖北武汉430070

出　　处：《南京农业大学学报》2023年第2期237-246,共10页Journal of Nanjing Agricultural University

基　　金：国家自然科学基金项目(31772289,31801843);广州市科技计划项目-科学研究重点项目(201904020033);广东省农产品保鲜物流共性关键技术研发创新团队项目(2021KJ145)。

摘　　要：[目的]本文旨在鉴定香蕉果实后熟关键调控因子MaMADS2的互作蛋白,深入解析MaMADS2影响香蕉果实后熟过程的分子机制。[方法]以MaMADS2为诱饵利用酵母双杂交文库获得候选互作蛋白,通过克隆候选互作基因编码区进行点对点酵母双杂交试验来验证互作情况;利用转录组学分析互作蛋白相关基因在后熟过程中的表达情况;利用双分子荧光互补试验鉴定重要互作蛋白与MaMADS2的互作;最后用蛋白免疫印迹分析MaMADS2蛋白在香蕉后熟过程中的表达。[结果]成功构建了果实在乙烯利催熟下的cDNA文库,初级和次级文库的库容分别为2.9×10^(6)和3.2×10^(6)CFU·mL^(-1),平均插入片段大小在1200 bp以上,可满足后续酵母双杂交筛选的要求。最终共鉴定出7个互作蛋白,其中有2个MADS-box转录因子,3个E3泛素连接酶,2个具有转录抑制作用的蛋白。转录组数据表明,乙烯利可抑制2个MADS-box基因的表达,而1-甲基环丙烯(1-MCP)对其表达起诱导作用。双分子荧光互补试验表明,MaMADS2与MADS-box转录因子Ma04_p30020.1存在互作关系。蛋白免疫印迹分析显示,MaMADS2在后熟过程中蛋白水平逐渐降低,这与其可以和E3泛素连接酶互作是一致的。[结论]通过构建高质量的果实cDNA文库与酵母双杂交筛库技术,鉴定出多个与MaMADS2互作的蛋白,并明确了MaMADS2与另一MADS-box转录因子Ma04_p30020.1之间存在明显的互作关系。[Objectives]The aim of this study was to identify the interacting proteins of MaMADS2,a key regulator of banana fruit ripening,and reveal the molecular mechanism of MaMADS2 regulating fruit ripening.[Methods]Using MaMADS2 as a“bait”,the candidate MaMADS2-interacting proteins were identified by yeast two-hybrid library,and its coding sequence of interacting proteins was cloned for confirming its interaction by one-to-one yeast two-hybrid assay.The expression of interacting protein related genes during fruit ripening was analyzed by transcriptome.The physical interactions between important interacting proteins and MaMADS2 were identified by bimolecular fluorescent complimentary(BiFC)assay.Finally,the expression of MaMADS2 protein during fruit ripening was analyzed by Western-blot assay.[Results]A cDNA library was successfully constructed with ethephon-treated banana fruit as materials.The capacities of the primary and secondary libraries were 2.9×10^(6) and 3.0×10^(6) CFU·mL^(-1),respectively,and the average size of inserts was above 1200 bp in the cDNA library,which could meet the requirements of subsequent yeast two-hybrid screening.Finally seven interacting proteins were determined,including two MADS-box transcription factor homologs,three E3 ubiquitin ligases,and two proteins with transcriptional inhibitory activity.Transcriptome data indicated that ethephon inhibited the expression of two MADS-box genes,while 1-MCP induced their expression.BiFC assay results showed that MaMADS2 and MADS-box transcription factor Ma04_P30020.1 could interact with each other.Moreover,Western-blot assay analysis indicated that the protein level of MaMADS2 decreased gradually during fruit ripening,which was consistent with the fact that E3 ubiquitin ligases could interact with it.[Conclusions]By constructing high-quality fruit cDNA library and yeast two-hybrid screening library technology,several proteins interacting with MaMADS2 were identified,and the interaction between MaMADS2 and another MADS-box transcription factor Ma

关 键 词：香蕉 酵母双杂交文库 MaMADS2 互作蛋白 果实成熟 

分 类 号：S667.9[农业科学—果树学]