山药DoWRKY40基因表达特征分析及互作蛋白筛选  

Analysis of DoWRKY40 Gene Expression Characteristics and Screening of Interacting Proteins in Yam

作　　者：邢丽南 张艳芳[1] 葛明然 赵令敏 陈妍 霍秀文[1] XING Li-nan;ZHANG Yan-fang;GE Ming-ran;ZHAO Ling-min;CHEN Yan;HUO Xiu-wen(College of Horticulture and Plant Protection,Inner Mongolia Agricultural University,Inner Mongolia Key Laboratory of Wild Endemic Vegetable Germplasm Resources and Germplasm Innovation,Hohhot 010019)

机构地区：[1]内蒙古农业大学园艺与植物保护学院,内蒙古自治区野生特有蔬菜种质资源与种质创新重点实验室,呼和浩特010019

出　　处：《生物技术通报》2024年第8期118-128,共11页Biotechnology Bulletin

基　　金：国家自然科学基金项目(32260759);内蒙古自然科学基金项目(2022MS03052);内蒙古自治区直属高校基本科研业务费项目(种业振兴领军人才专项基金)(BR22-11-06)。

摘　　要：【目的】WRKY作为植物特异型转录因子,参与植物生长发育过程。克隆山药DoWRKY40基因,分析其表达模式,通过酵母双杂交技术筛选与DoWRKY40互作的蛋白,探究WRKY40在山药膨大过程中的调控机制。【方法】以‘大和长芋’山药为材料克隆DoWRKY40基因,并进行生物信息学分析、表达模式分析、亚细胞定位,构建山药块茎不同发育时期酵母文库,筛选与DoWRKY40互作蛋白。【结果】DoWRKY40的开放阅读框长为927 bp。DoWRKY40蛋白属于WRKY家族的第II组,含有一个典型的WRKY转录因子保守结构域,定位于细胞核。与几内亚薯蓣亲缘关系较近。DoWRKY40基因在山药块茎生长发育的120 d表达量最高,且在叶、茎和块茎中均有表达,具有组织特异性。山药cDNA文库的库容量为1.484×10^(8);pGBKT7-WRKY40诱饵载体无自激活活性。酵母双杂交法从山药文库中筛选到4类与DoWRKY40相互作用的蛋白,分别参与植物生长发育、细胞周期调节与细胞扩增、信号转导及环境胁迫应答等。【结论】克隆得到DoWRKY40基因,其响应山药的生长发育过程,筛选到的4类互作蛋白表明其在山药细胞膨大及信号转导中起调控作用。【Objective】WRKY,as a plant idiosyncratic transcription factor,is involved in plant growth and development.DoWRKY40 gene in yam(Dioscorea opposita Thunb.)was cloned and its expression pattern was analyzed.The protein interacting with DoWRKY40 was screened by yeast two-hybrid technique to explore the regulatory mechanism of WRKY40 in the process of yam expansion.【Method】DoWRKY40 gene was cloned from‘Dahechangyu’yam,and bioinformatics analysis,expression pattern analysis and subcellular localization were performed.Yeast libraries of yam tuber at different developmental stages were constructed and DoWRKY40 interacting proteins were screened.【Result】The length of open reading frame of DoWRKY40 was 927 bp;DoWRKY40 belonged to group II of the WRKY family and contained a typical WRKY transcription factor conserved domain,which was localized in the nucleus.It was closely related to Dioscorea cayenensis subsp.The expression of DoWRKY40 gene was the highest at 120 d after the growth and development of yam tuber,and it was expressed in the leaves,stems and tubers with tissue specificity.The library capacity of yam cDNA library was 1.484×10^(8);pGBKT7-WRKY40 decoy vector had no self-activation activity.Four kinds of proteins interacting with DoWRKY40 were screened from yam library by yeast two-hybridization,which were involved in plant growth and development,cell cycle regulation and cell expansion,signal transduction and environmental stress response,respectively.【Conclusion】The DoWRKY40 gene is cloned,which responds to the growth and development process of yam.The four interacting proteins are screened,suggesting that it plays a regulatory role in the expansion and signal transduction of yam cells.

关 键 词：山药 WRKY40基因 表达特征 亚细胞定位 CDNA文库 酵母双杂交 互作蛋白筛选 

分 类 号：S632.1[农业科学—蔬菜学] Q943.2[农业科学—园艺学]