甘薯IbKFB互作蛋白的筛选及分析  

Screening and analysis of IbKFB interacting protein in sweetpotato

作　　者：常璐 董海涛 赵彩良 张红梅[3] 武小平[3] 吕运韬 贾小云[1] 唐锐敏 贺立恒[2] CHANG Lu;DONG Haitao;ZHAO Cailiang;ZHANG Hongmei;WU Xiaoping;LU Yuntao;JIA Xiaoyun;TANG Ruimin;HE Liheng(College of Life Sciences,Shanxi Agricultural University,Jinzhong,Shanxi 030801,China;College of Agronomy,Shanxi Agricultural University,Jinzhong,Shanxi 030801,China;Maize Research Institute,Shanxi Agricultural University,Xinzhou,Shanxi 034000,China)

机构地区：[1]山西农业大学生命科学学院,山西晋中030801 [2]山西农业大学农学院,山西晋中030801 [3]山西农业大学玉米研究所,山西忻州034000

出　　处：《植物生理学报》2023年第10期1890-1898,共9页Plant Physiology Journal

基　　金：国家青年科学基金(31900450);山西省农业科学院应用基础研究计划(YGC2019FZ4);中央引导地方科技发展资金项目(YDZX-20201400001019);吕梁市引进高层次科技人才重点研发项目(2021RC-2-21);国家外国专家局高端专家引智项目(G2022004007L和G2023004003L);山西省回国留学人员科研资助项目(2022-094);忻州市重点研发计划-农业领域(202102025)。

摘　　要：甘薯(Ipomoea batatas)是一种极为重要的粮食、饲料和工业原料类块根作物。与普通甘薯相比,紫心甘薯块根中富含花青素,已成为当前功能性食品开发的热点作物。本课题组前期通过不同薯肉颜色的甘薯块根的小RNA、降解组和转录组测序以及靶向代谢组联合分析发现,Ib-miRNA2111及其候选靶基因(IbKFB)可能参与花青素生物合成调控。为了深入探究IbKFB在甘薯花青素生物合成过程中的功能和调控机理,本研究克隆了IbKFB基因,构建IbKFB的酵母双杂诱饵载体pGBKT7-IbKFB,采用酵母双杂交(Y2H)技术从甘薯cDNA文库中筛选出21个与IbKFB相互作用的候选蛋白。通过对这些蛋白进行功能注释和分析,鉴定出5个注释与花青素生物合成相关的候选蛋白,分别为IbPAL、IbSAUR50、IbERF、IbWDR11和IbGAPCp1。进一步利用Y2H回转实验和双分子荧光互补实验(BiFC)验证了IbKFB与候选蛋白相互作用的真实性。本研究可丰富甘薯花青素生物合成的分子调控网络和植物地下器官花青素代谢调控机制的认识,为花青素生物强化专用型甘薯新种质的创制提供分子理论基础和基因资源。Sweetpotato(Ipomoea batatas)is a highly valued root crop used for food,feed and industrial raw material.Compared with ordinary sweetpotato,purple-fleshed sweetpotato storage roots are abundant in anthocyanins,which has become a hot crop in functional food development.In our previous study,through the combined analysis of small RNA,degradation group,transcriptomic sequencing and targeted metabolome of sweetpotato root storage roots with different flesh colors,we discovered that Ib-miRNA2111 and its candidate target gene(IbKFB)may be involved in regulating anthocyanin biosynthesis.In order to further explore the function and regulatory mechanism of IbKFB in anthocyanin biosynthesis of sweetpotato,we cloned the Ib KFB gene and constructed the yeast decoy vector pGBKT7-IbKFB.Using yeast two-hybridization(Y2H),we screened 21 candidate proteins from sweetpotato cDNA library that interacted with IbKFB.By functional annotation and analysis of these proteins,we identified five candidate proteins:IbPAL,Ib SAUR50,IbERF,IbWDR11 and IbGAPCp1.Further validation of the interaction between IbKFB and candidate proteins was confirmed through Y2H reversion assay and bimolecular fluorescence complementation(Bi FC)analysis.This study improves our understanding of the molecular regulatory network of anthocyanin biosynthesis in sweetpotato,as well as the regulation mechanism of anthocyanin metabolism in underground plant organs.Furthermore,it provides a molecular theoretical basis and gene resources for the development of new sweetpotato varieties with enhanced anthocyanin content.

关 键 词：甘薯 花青素 IbKFB 互作蛋白 

分 类 号：S531[农业科学—作物学]