玉米 ZmENA1 基因的克隆及其对金属离子的应答分析

doi:10.13560/j.cnki.biotech.bull.1985.2020-0192

摘要/Abstract

摘要：

烟酰胺（NA）是重要的植物铁载体麦根酸（MA）的前体物质,烟酰胺外排转运体 ENA 是植物体介导 NA 转运的重要蛋白。探究 ZmENA1 的基本功能可为阐明玉米中金属离子的转运及储存机制提供依据。本研究同源克隆了 ZmENA1 基因,并通过生物信息学方法分析了该蛋白的基本特征,同时运用实时荧光定量技术分析了该基因在籽粒发育过程及在不同金属离子浓度下的表达模式。结果表明,ZmENA1 的 ORF 长 1 356 bp,预测其编码蛋白等电点为 8.06,蛋白质大小为 48.998 kD,在蛋白质二级结构中 α 螺旋占比达 53.98%;该蛋白有 4 个磷酸化位点、10 个跨膜区且 N 端含有 1 个信号肽。表达分析发现,ZmENA1 在胚乳和种皮混合物中的表达量高于胚,并在 16 DAP达到高峰,然后随着发育进程逐渐降低。此外,ZmENA1 的表达可应答于 Zn²⁺、Mn²⁺ 和 Cu²⁺ 的缺乏以及过量的 Zn²⁺、Mn²⁺ 和 Cd²⁺,推测 ZmENA1 在调节金属离子的转运和储藏方面发挥作用。本研究为深入揭示 ZmENA1 的功能提供基础。

关键词: 玉米, ZmENA1, 金属, 烟酰胺, 生物信息学

Abstract:

Nicotianamine（NA）is the precursor of mugineic acid（MA）which is a phytosiderophore of plants,and nicotianamine effluxer（ENA）is an important protein mediating NA transport in plants. Thus,exploring the functions of ZmENA1 may provide bases for understanding metal ions transport and storage mechanisms in maize. In this study,ZmENA1 was homologously cloned and the basic characteristics of the encoded protein were analyzed via bioinformatics. Also the expression patterns of ZmENA1 during kernel development process under different metal ions concentrations were examined using real-time quantitative PCR. The results showed that the ORF of ZmENA1 was 1 356 bp,and the encoded protein had a deduced isoelectric point of 8.06 and molecular weight of 48.998 kD. In addition,ZmENA1 contained 53.98% proportion of α-helix,four phosphorylation sites,10 transmembrane regions,and 1 N terminal signal peptide. The expression of ZmENA1 in the mixture of endosperm and pericarp was higher than that in the embryo,reached a peak at 16 DAP,and decreased with the developmental process. Besides,the expression of ZmENA1 presented fluctuations in response to deficiency of Zn²⁺,Mn²⁺,and Cu²⁺,as well as excess of Zn²⁺,Mn²⁺,and Cd²⁺,suggesting that ZmENA1 played a role in mediating transport and storage of these metal ions. This study may provide a basis for further revealing the function of ZmENA1.

Key words: maize, ZmENA1, metal, nicotianamine, bioinformatics

张鑫, 周晓今, 逄森, 李素贞, 黄家兴, 陈茹梅. 玉米 ZmENA1 基因的克隆及其对金属离子的应答分析[J]. 生物技术通报, 2020, 36(11): 1-8.

ZHANG Xin, ZHOU Xiao-jin, PANG Sen, LI Su-zhen, HUANG Jia-xing, CHEN Ru-mei. Cloning of ZmENA1 and Its Responses to Metal Ions in Maize[J]. Biotechnology Bulletin, 2020, 36(11): 1-8.

图/表 6

图1 ZmENA1基因的克隆

图2 ZmENA1 蛋白的二级和三级结构预测 A：ZmENA1 蛋白二级结构预测;B：ZmENA1 蛋白三级结构预测

图3 ZmENA1 蛋白磷酸化位点预测

图4 ZmENA1 蛋白跨膜区和信号肽预测 A：ZmENA1 蛋白跨膜区预测;B：ZmENA1 蛋白信号肽预测

图5 ZmENA1 在籽粒发育过程中的表达 Seed：籽粒;Em：胚;En：胚乳和种皮混合物;利用玉米的ZmActin1基因为内参,计算该基因的相对表达量,并通过2-ΔΔCt的方法计算基因的表达量;误差线表示标准偏差

图6 ZmENA1对不同浓度金属元素的响应表达 CK：对照,正常营养液;++：高浓度金属营养液;--：缺素营养液;Root：幼苗地下部分;Shoot：幼苗地上部分;利用玉米的ZmActin1基因为内参,计算该基因的相对表达量,并通过2-ΔΔCt的方法计算基因的表达量;误差线表示标准偏差;星号代表的是显著差异,*代表P<0.05,**代表P<0.01,***代表P<0.001

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