‘白花建’莲酵母双杂文库构建及NnWRKY40互作蛋白筛选  

Construction of two-hybrid library of yeast and screening of NnWRKY40 interacting proteins in Nelumbo nucifera Gaertn.‘Baihuajian’

作　　者：陈思梦 王锦鑫 党明静 王政道 李静 Chen Si-Meng;Wang Jin-Xin;Dang Ming-Jing;Wang Zheng-Dao;Li Jing(School of Chemistry,Chemical Engineering and Life Sciences,Wuhan University of Technology,Wuhan 430070,China)

机构地区：[1]武汉理工大学化学化工与生命科学学院生物技术系,武汉430070

出　　处：《植物科学学报》2023年第4期447-457,共11页Plant Science Journal

基　　金：国家自然科学基金项目(31700262);山东省自然科学基金项目(ZR2021MC163)。

摘　　要：通过构建莲(Nelumbo nucifera Gaertn.)品种‘白花建’不同组织的混合cDNA文库,筛选与莲转录因子NnWRKY40互作的蛋白,探究NnWRKY40参与调控次级代谢物生物碱合成的可能机制。结果显示,混合cDNA文库的库容为1.2×107 CFU,重组率为100%,插入片段平均长度大于1000 bp。NnWRKY40包含两个同源基因NnWRKY40a和NnWRKY40b,利用NnWRKY40b构建诱饵载体pGBKT7-NnWRKY40b,通过共转化方法,从文库中筛选到27个与NnWRKY40b互作的蛋白。这些互作蛋白可分为生长发育及抗逆、激素调控和次级代谢、未知蛋白3类。选取6个代表性互作蛋白(NnUBC、NnPEBP、NnPPOA、NnCHS、NnJAZ1和Unknown protein 3)进行一对一验证,发现其中JAZ蛋白与次生代谢物生物碱合成相关,提示NnWRKY40b转录因子可能与茉莉酸(JA)介导的生物碱合成调控密切相关。To explore the potential mechanism of the NnWRKY40 transcription factor in lotus(Nelumbo nucifera Gaertn.)for regulating the synthesis of secondary metabolite alkaloids,a mixed cDNA library from different lotus tissues was constructed,and the proteins interacting with NnWRKY40 were screened.Total RNA of different tissues was extracted from‘Baihuajian’,and a mixed cDNA library was established.The library capacity was 1.2×107 CFU,recombinant rate was 100%,and average length of the inserted fragments was>1000 bp.NnWRKY40 contains two homologous genes,NnWRKY40a and NnWRKY40b.As NnWRKY40b is reported to play a leading role in transcriptional activation of alkaloid synthesis genes,we used NnWRKY40b to construct the bait vector PGBKT7-NnWRKY40b.In total,27 proteins interacting with NnWRKY40b were screened from the library using the co-transformation method.These interacting proteins could be divided into three categories:i.e.,growth and development and stress response,hormone regulation and secondary metabolism,and unknown proteins.Six representative proteins,including NnUBC,NnPEBP,NnPPOA,NnCHS,NnJAZ1,and unknown protein 3,were selected for one-to-one verification,among which the JAZ pro-tein was associated with alkaloid synthesis,suggesting that the NnWRKY40b transcription factor may be closely related to jasmonic acid(JA)-mediated regulation of alkaloid synthesis in N.nucifera.

关 键 词：莲 酵母双杂交 NnWRKY40 蛋白互作 

分 类 号：Q943.2[生物学—植物学]