木薯MeAHL19的互作蛋白筛选及鉴定  

Screening and Identification of Interaction Proteins of MeAHL19 in Cassava(Manihot esculenta)

作　　者：张建禹 王晓彤 耿沙 毋志浩 任思杨 李瑞梅[2] 姚远[2] 郭建春[2] 刘姣[2] 胡新文[1] ZHANG Jian-Yu;WANG Xiao-Tong;GENG Sha;WU Zhi-Hao;REN Si-Yang;LI Rui-Mei;YAO Yuan;GUO Jian-Chun;LIU Jiao;HU Xin-Wen(School of Life Sciences,Hainan University,Haikou 570228,China;Institute of Tropical Bioscience and Biotechnology,Chinese Academy of Tropical Agricultural Sciences/Hainan Institute for Tropical Agricultural,Haikou 571101,China;College of Tropical Crops,Hainan University,Haikou 570228,China)

机构地区：[1]海南大学生命科学学院,海口570228 [2]中国热带农业科学院热带生物技术研究所/海南热带农业资源研究院,海口571101 [3]海南大学热带作物学院,海口570228

出　　处：《农业生物技术学报》2022年第8期1454-1467,共14页Journal of Agricultural Biotechnology

基　　金：国家重点研发项目(2019YFD1001105);国家自然科学基金(32001602);国家现代农业产业技术体系(CARS-11)。

摘　　要：AT-hook核定位蛋白(AT-hook motif nuclear localized protein,AHL)是一类具有AT-hook基序的核定位蛋白,在植物生长发育过程中起着重要的调控作用。为探究木薯(Manihot esculenta)AHL家族成员MeAHL19的生物学功能以及其在蛋白互作网络中的作用。本研究采用T4连接法构建诱饵载体pGBKT7-MeAHL19,利用酵母(Saccharomyces cerevisiae)双杂交技术(yeast two-hybrid,Y2H)在木薯cDNA文库中筛选MeAHL19的互作蛋白,采用实时荧光定量PCR技术分析MeAHL19及其互作蛋白的基因表达模式。结果显示:酵母双杂交筛库获得20个阳性克隆,经PCR扩增并测序比对后鉴定出8个候选互作蛋白,Y2H回转验证发现,MeAHL19蛋白与核酮糖二磷酸羧化酶小链(ribulose bisphosphate carboxylase small chain,RBCS)蛋白可在体外互作;双分子荧光互补(bimolecular fluorescence complementation,BiFC)实验进一步验证了MeAHL19与RBCS在本氏烟草(Nocotinan benthamiana)叶肉细胞中发生互作。基因表达模式分析表明,MeAHL19主要在根中表达,而RBCS在根、茎和叶中的表达水平基本一致;MeAHL19与RBCS在木薯中共同响应非生物胁迫和外源激素应答。本研究为进一步探究MeAHL19在木薯逆境响应过程的生物学功能提供了科学参考。The AT-hook motif nuclear localized protein(AHL)family genes contain a unique combination of1 or 2 AT-hook domains and regulate various biological processes in plants.To explore the biological function of cassava(Manihot esculenta)AHL family gene MeAHL19 and its role in the protein interaction network,in this study,the bait vector pGBKT7-MeAHL19 was constructed using T4 ligase and used to screen the cassava cDNA library via the yeast(Saccharomyces cerevisiae)two-hybrid(Y2H)library screening system.Real-time quantitative PCR method was used to detect gene expression patterns of MeAHL19 and the interacting proteins.The results showed that 20 positive clones were obtained through Y2H screening.Eight candidate interacting proteins were identified by PCR amplification and sequencing alignment.Y2H test confirmed the interaction between MeAHL19 and ribulose bisphosphate carboxylase small chain(RBCS)protein in vitro.BiFC results further confirmed that MeAHL19 interacted with RBCS in Nicotiana benthamiana mesophyll cells.Analysis of gene expression patterns showed that MeAHL19 was mainly expressed in roots,whereas RBCS was expressed at about the same level in roots,stems and leaves;MeAHL19 and RBCS responded to abiotic stress and exogenous hormone responses in cassava.This study provides a reference for further investigation on the biological function of MeAHL19 in stress responses of cassava.

关 键 词：木薯 木薯AT-hook核定位蛋白19(MeAHL19) 酵母双杂交 互作蛋白 双分子荧光互补 

分 类 号：S533[农业科学—作物学]