玉米ZmSTP1和ZmAAP2基因启动子区域结合蛋白的检测  

Exploration of binding proteins of ZmSTP1 and ZmAAP2 in maize

作　　者：余娇娇 赵雯 肖池贵 王婷 马兰 陶增 YU Jiao-jiao;ZHAO Wen;XIAO Chi-gui;WANG Ting;MA Lan;TAO Zeng(Institute of Biology and Environmental Engineering,School of Chemical Biology and Environment,Yuxi Normal Universtiy,Yuxi,Yunnan 653100,China)

机构地区：[1]玉溪师范学院化学生物与环境学院/生物与环境工程研究院,云南玉溪653100

出　　处：《西南农业学报》2023年第5期897-903,共7页Southwest China Journal of Agricultural Sciences

基　　金：国家自然科学基金地区科学基金项目(32160741);云南省地方本科高校基础研究联合专项面上项目(2018FH001-041);云南省大学生创新创业训练计划项目(2021A072)。

摘　　要：【目的】植物特异性转录因子NLPs(NIN-like proteins)是硝酸盐响应顺式元件(NRE)的结合蛋白,在硝酸盐调控的下游基因表达中起转录激活作用。玉米转运蛋白ZmSTP1和ZmAAP2在植物碳氮产物的运输和卸载过程中发挥作用,且基因启动子区域均含有一个硝酸盐响应顺式元件。本研究旨在证实玉米ZmNLPs家族成员ZmNLP3、ZmNLP4是否为ZmSTP1和ZmAAP2基因启动子中NRE的结合蛋白。【方法】以玉米B73为试验材料,首次利用酵母单杂交技术检测ZmNLP3、ZmNLP4转录因子分别与ZmSTP1和ZmAAP2基因启动子区域的相互作用。【结果】在线预测网站Plantcare对ZmSTP1和ZmAAP2基因转录起始位点上游2000 bp序列分析表明,位于2个基因转录起始点上游-577~-589 bp和-909~-921 bp区域均包含一个序列长12 bp的NRE元件,结构分别为5′-ATTTAATACTCA-3′和5′-ATATATAAGTCA-3′;诱饵菌株AbAr基因表达检测显示,能抑制诱饵菌株Y1H(pAbAi-ZmSTP1)和Y1H(pAbAi-ZmAAP2)本底表达的最低AbA浓度均为200 ng/mL;将pGADT7-ZmNLP3/4分别转化Y1H(pAbAi-ZmSTP1)和Y1H(pAbAi-ZmAAP2)菌株后,在对照培养基(SD/-Leu)和含AbA浓度为200 ng/mL的筛选培养基中Y1H[(pAbAi-ZmSTP1)/(pGADT7-ZmNLP3)]、Y1H[(pAbAi-ZmSTP1)/(pGADT7-ZmNLP4)]、Y1H[(pAbAi-ZmAAP2)/(pGADT7-ZmNLP3)]、Y1H[(pAbAi-ZmAAP2)/(pGADT7-ZmNLP4)]菌株均能正常生长。【结论】玉米转录因子ZmNLP3、ZmNLP4均能与ZmSTP1和ZmAAP2基因启动子区域相互作用,是靶基因ZmSTP1和ZmAAP2启动子区域NRE的结合蛋白。本研究为深入了解ZmNLPs家族成员参与的硝酸盐信号响应提供理论基础,也为玉米增产和氮素增效提供分子标记和理论依据。The NLPs family of transcription factors were identified as nitrate-responsive ciselement(NRE)-binding proteins, which functioned as transcriptional activators in the nitrate-regulated expression of downstream genes. Transporters of ZmSTP1 and ZmAAP2 played important roles in the transport and unloading of C-N assimilates in maize. In addition, both ZmSTP1 and ZmAAP2 contained NRE cis-elements in the promoter regions. The present study aimed to determine whether NLP gene family in maize were NRE binding proteins of ZmSTP1 and ZmAAP2 promoters. 【Method】Maize B73 as experimental material was used to explore the interaction between ZmNLP3,ZmNLP4,ZmSTP1 and ZmAAP2 by yeast one-hybrid system.【Result】The analysis of 2000 bp sequences upstream of the transcription start sites of ZmSTP1 and ZmAAP2 genes by Plantcare showed that NRE cis-elements were located in-577 to-589 bp and-909 to-921 bp regions upstream of the transcription start sites of the two genes. The structures were 5′-ATTTAATACTCA-3′and 5′-ATATATAAGTCA-3′.AbA~r gene expression detection of bait strains showed that the lowest AbA concentration that could inhibit the background expression of bait strains Y1H(pAbAi-ZmSTP1) and Y1H(pAbAi-ZmAAP2)was 200 ng/mL. The pGADT7-ZmNLP3/4 were transformed into the Y1H(pAbAi-ZmSTP1) and Y1H(pAbAi-ZmAAP2) strains, and all strains could grow normally on control medium(SD/-Leu) and screening medium with AbA concentration of 200 ng/mL.【Conclusion】ZmNLP3 and ZmNLP4 can interact with ZmSTP1 and ZmAAP2.ZmNLP3 and ZmNLP4 are binding proteins of NRE elements in the promoter region of ZmSTP1 and ZmAAP2. The results of this study not only contribute to the further understanding of nitrate signal transduction involving members of ZmNLPs family, but also provide the theory basis for molecular breeding for increasing maize yield and nitrogen efficiency.

关 键 词：玉米 酵母单杂交 转运蛋白 ZmNLPs 

分 类 号：S513[农业科学—作物学]