玉米ZmNLP5与ZmSTP1、ZmAAP2基因启动子区域相互作用的鉴定

doi:10.3969/j.issn.1004-1524.2022.11.03

浙江农业学报 ›› 2022, Vol. 34 ›› Issue (11): 2340-2347.DOI: 10.3969/j.issn.1004-1524.2022.11.03

玉米ZmNLP5与ZmSTP1、ZmAAP2基因启动子区域相互作用的鉴定

袁崇渊(), 祝愿飞, 陈霞, 朱婵, 王毅, 陶海燕, 余娇娇()

玉溪师范学院化学生物与环境学院, 云南玉溪 653100

收稿日期:2022-05-06 出版日期:2022-11-25 发布日期:2022-11-29
作者简介:*余娇娇,E-mail: jiaojiaoyu@yxnu.edu.cn
袁崇渊(2000—),女,云南德宏人,本科生,主要从事玉米营养生理与遗传研究。E-mail: 962573436@qq.com
通讯作者: 余娇娇
基金资助:
国家自然科学基金(32160741);云南省地方本科高校基础研究联合专项(2018FH001-041);云南省大学生创新创业训练计划(202011390029)

Identification of the interaction between ZmNLP5 and promoters of ZmSTP1, ZmAAP2 gene in maize

YUAN Chongyuan(), ZHU Yuanfei, CHEN Xia, ZHU Chan, WANG Yi, TAO Haiyan, YU Jiaojiao()

School of Chemical Biology and Environment, Yuxi Normal Universtiy, Yuxi 653100, Yunnan,China

Received:2022-05-06 Online:2022-11-25 Published:2022-11-29
Contact: YU Jiaojiao

摘要/Abstract

摘要：

氮(N)是控制玉米产量的主要限制性因素,玉米对氮素的利用效率可以通过增加碳(C)的有效性得以改善。玉米转运蛋白ZmSTP1和ZmAAP2在植物C/N产物的运输和卸载过程中起着重要作用,且两个基因的启动子区域均含有一个硝酸盐响应顺式元件(nitrate responsive cis element,NRE)。NIN-like protein(NLP)是一类保守的植物特异性转录因子,已在多种植物被证实在调控N响应中发挥关键作用,其中玉米ZmNLP5是介导氮信号转导和代谢分子网络的中枢基因之一。为了明确ZmNLP5是否能与ZmSTP1和ZmAAP2基因启动子区域的NRE结合,以玉米B73为实验材料,首次利用酵母单杂交技术检测ZmNLP5与ZmSTP1、ZmAAP2基因启动子区域的相互作用。结果表明,将pGADT7-ZmNLP5-1/2分别转化Y1H (pAbAi-ZmSTP1)和Y1H(pAbAi-ZmAAP2)菌株后,在SD/-Leu培养基上均有菌落生长,在含AbA抗生素浓度为200 ng·mL^-1的SD/-Leu培养基中只有Y1H(pAbAi-ZmAAP2/pGADT7-ZmNLP5-1 )菌株能正常生长。结果说明,ZmNLP5只与ZmAAP2基因启动子区域相互作用,ZmNLP5是能与ZmAAP2基因启动子NRE结合的转录因子。本研究结果不仅有助于进一步了解玉米硝酸盐信号转导的调控途径,也为今后其他植物硝酸盐信号转导的研究提供理论依据。

关键词: 玉米, 酵母单杂交, 转运蛋白

Abstract:

Nitrogen (N) is the major limitation factor for its grain production, and both genes contain a NRE cis element in the promoter region.N use efficiency of maize may be improved by increasing the availability of carbon (C). Transporters of ZmSTP1 and ZmAPP2 play important roles in the transport and unloading of C-N assimilates in maize. In addition, both ZmSTP1 and ZmAAP2 contain NRE cis elements in promoter regions. NIN-like protein (NLP) is a conserved plant-specific transcription factor family and has been shown in several plant species to be a key player in regulating nitrogen response. ZmNLP5 is a central hub in a molecular network for mediating N signalling and metabolism. To understand whether ZmNLP5 is involved in regulating of ZmSTP1 and ZmAAP2, we used maize B73 as experimental material to explore the interaction between ZmNLP5, ZmSTP1 and ZmAAP2 by yeast one-hybrid system. The results showed that the pGADT7-ZmNLP5-1/2 were transformed into the Y1H(pAbAi-ZmSTP1) and Y1H(pAbAi-ZmAAP2) strains, respectively, they all grew normally on control medium (SD/-Leu). On selection medium (SD/-Leu/+200 ng·mL^-1AbA), the growth of Y1H(pAbAi-ZmAAP2/pGADT7-ZmNLP5-1) strain had been in normal. This result suggested that ZmNLP5 can interact with ZmAAP2 gene, ZmNLP5 is a transcription factor that can bind to the NRE element of ZmAAP2. The results of this study not only contribute to the further understanding of the regulatory mechanism of nitrate signal transduction in maize, but also provide a theoretical basis for the future study of nitrate signal transduction in other plants.

Key words: maize, yeast one-hybrid, transport protein

中图分类号:

S513

袁崇渊, 祝愿飞, 陈霞, 朱婵, 王毅, 陶海燕, 余娇娇. 玉米ZmNLP5与ZmSTP1、ZmAAP2基因启动子区域相互作用的鉴定[J]. 浙江农业学报, 2022, 34(11): 2340-2347.

YUAN Chongyuan, ZHU Yuanfei, CHEN Xia, ZHU Chan, WANG Yi, TAO Haiyan, YU Jiaojiao. Identification of the interaction between ZmNLP5 and promoters of ZmSTP1, ZmAAP2 gene in maize[J]. Acta Agriculturae Zhejiangensis, 2022, 34(11): 2340-2347.

图/表 5

图1 ZmSTP1和ZmAAP2基因启动子中NRE位点示意图

Fig.1 Schematic diagram of nitrate response element of ZmSTP1 and ZmAAP2

表1 克隆引物序列

Table 1 The primer sequences for clone

基因 Gene	上游引物序列 Forward primer sequences(5'→3')	下游引物序列 Reverse primer sequences(5'→3')
ZmSTP1	CTGTAGAGCGTAAAACTAATATATCA	AAAATGACTAGCATTTTGGGATGG
ZmAAP2	TTGTTTGATCTTGATTGATGAAGTAATAA	ATAGAAAATAGCATAAGATATTCCAAGTAC
ZmNLP5-1	CTGAACGGTCATGGAGGAGAC	CTTGGCGAGCTTGCGGAAC
ZmNLP5-2	AGAGTGGCCTCCTCCCAAG	CTCCTGGAAGTCGGCGTC

图2 诱饵载体线性化产物琼脂糖凝胶电泳图 M, 10 000 plus DNA marker; 1,未经酶切的pAbAi-ZmAAP2质粒;2,单酶切后的pAbAi-ZmAAP2质粒;3,未经酶切的pAbAi-ZmSTP1质粒;4,单酶切后的pAbAi-ZmSTP1质粒。

Fig.2 Agarose gel electrophoresis image of the linearized products of bait M, 10 000 plus DNA marker;1, pAbAi-ZmAAP2 plasmid without enzyme digestion; 2, pAbAi-ZmAAP2 plasmid after single enzyme digestion; 3, pAbAi-ZmSTP1 plasmid without enzyme digestion; 4, pAbAi-ZmSTP1 plasmid after single enzyme digestion.

图3 诱饵酵母AbAr背景表达水平检测 A,诱饵菌株Y1H(pAbAi-ZmSTP1);B,诱饵菌株Y1H(pAbAi-ZmAAP2)。

Fig.3 Detection of AbAr expression level in bait yeast strain A, Bait strain of Y1H(pAbAi-ZmSTP1); B, Bait strain of Y1H(pAbAi-ZmAAP2).

图4 酵母单杂检测诱饵DNA和猎物蛋白的相互作用 A,阴性对照Y1H(AbAi-P53/pGADT7);B,阳性对照Y1H(AbAi-P53/pGADT7-P53);C,Y1H(pAbAi-ZmSTP1/pGADT7-ZmNLP5-1)相互作用图;D,Y1H(pAbAi-ZmSTP1/pGADT7-ZmNLP5-2)相互作用图;E,Y1H(pAbAi-ZmAAP2/pGADT7-ZmNLP5-1)相互作用图;F,Y1H(pAbAi-ZmAAP2/pGADT7-ZmNLP5-2)相互作用图。

Fig.4 Interaction between bait DNA and prey protein was detected by yeast one-hybrid system A, Negative control of Y1H(AbAi-P53/pGADT7); B, Positive control of Y1H(AbAi-P53/pGADT7-P53); C, The interaction of pGADT7-ZmNLP5-1and Y1H(pAbAi-ZmSTP1); D, The interaction of pGADT7-ZmNLP5-2 and Y1H(pAbAi-ZmSTP1); E, The interaction of pGADT7-ZmNLP5-1 and Y1H(pAbAi-ZmAAP2); F, The interaction of pGADT7-ZmNLP5-2 and Y1H(pAbAi-ZmAAP2).

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玉米ZmNLP5与ZmSTP1、ZmAAP2基因启动子区域相互作用的鉴定

Identification of the interaction between ZmNLP5 and promoters of ZmSTP1, ZmAAP2 gene in maize

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