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作物学报 ›› 2020, Vol. 46 ›› Issue (8): 1185-1194.doi: 10.3724/SP.J.1006.2020.93062

• 作物遗传育种·种质资源·分子遗传学 • 上一篇    下一篇

玉米阳离子/质子逆向转运蛋白ZmNHX7的功能鉴定

张凌霄1,2,焦珍珍2,卜华虎2,王逸茹2,李健2,3,郑军1,2,*()   

  1. 1北京农学院植物科学技术学院, 北京 102206
    2中国农业科学院作物科学研究所, 北京 100081
    3吉林农业大学农学院, 吉林长春 130118
  • 收稿日期:2020-02-06 接受日期:2020-03-24 出版日期:2020-08-12 网络出版日期:2020-04-03
  • 通讯作者: 郑军
  • 作者简介:E-mail: 13522840682@163.com
  • 基金资助:
    国家重点研发计划项目(2016YFD0101002)

Functional identification of maize cation/proton antiporter ZmNHX7

ZHANG Ling-Xiao1,2,JIAO Zhen-Zhen2,BU Hua-Hu2,WANG Yi-Ru2,LI Jian2,3,ZHENG Jun1,2,*()   

  1. 1College of Plant Science and Technology, Beijing University of Agriculture, Beijing 102206, China
    2Institute of Crop Sciences, Chinese Academy of Agricultural Sciences, Beijing 100081, China
    3College of Agronomy, Jilin Agricultural University, Changchun 130118, Jilin, China
  • Received:2020-02-06 Accepted:2020-03-24 Published:2020-08-12 Published online:2020-04-03
  • Contact: Jun ZHENG
  • Supported by:
    National Key Research and Development Program of China(2016YFD0101002)

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摘要：

植物阳离子/质子逆向转运蛋白可以维持细胞内的离子平衡, 在抵御离子毒害过程中发挥重要作用。本研究克隆了一个编码玉米阳离子/质子逆向转运蛋白的基因, 命名为ZmNHX7。该基因编码序列(coding sequence, CDS)全长3411 bp, 编码一条含1136个氨基酸的多肽链。ZmNHX7基因在玉米各组织部位均有表达, 在V7 (第7片叶完全展开)时期的根和茎中表达量较高。在NaCl与LiCl的胁迫条件下, 该基因表达量上调。系统进化树分析将ZmNHX7与拟南芥质膜阳离子/质子逆向转运蛋白AtNHX7和AtNHX8归为一类, 亚细胞定位结果表明该蛋白定位于细胞膜和核膜上。将ZmNHX7基因转入拟南芥T-DNA插入突变体中, 转基因互补株系可以恢复该突变体对Li+的耐受性。这些结果表明, ZmNHX7编码一个玉米质膜阳离子/质子逆向转运蛋白, 在缓解Li+对植物的毒害和维持细胞内的离子平衡等方面发挥重要作用。

关键词: 玉米, ZmNHX7, 逆向转运蛋白, 离子毒害

Abstract:

Plant cation/proton antiporters can maintain intracellular ion homeostasis and resist ion toxicity. In this study, a gene encoding a maize cation/proton antiporter was cloned and named as ZmNHX7. The coding sequence of ZmNHX7 was 3411 bp, encoding a protein with 1136 amino acids. ZmNHX7 is ubiquitously expressed in various tissues of maize, With higher expression level in roots and stems at the V7 stage. The expression of ZmNHX7 was induced by NaCl and LiCl stresses. In phylogenetic tree ZmNHX7 showed a close relation with AtNHX7 and AtNHX8 of Arabidopsis thaliana. ZmNHX7 was located in cell membrane and nuclear membrane by confocal laser scanning microscopy analysis of the lower epidermis of tobacco leaves. When ZmNHX7 gene was transformed into the Arabidopsis thaliana T-DNA insertion mutant nhx8, the transgenic complementary lines could restore the tolerance of nhx8 to Li+. These results indicate that ZmNHX7 encodes a plasma membrane cation/proton antiporter of maize, which plays an important role in reducing the toxicity of Li+ to plants and maintaining intracellular ion homeostasis.

Key words: maize, ZmNHX7, antiporter, ion toxicity

表1

引物序列"

引物用途Purpose of primer 引物名称Primer name 引物序列Primer sequence (5'-3')
扩增基因引物 ZmNHX7-G-F1 ATGGGCGGCGAGGCTGAGCC
Amplified gene primer sequences ZmNHX7-G-R1 CTACTGCTCCTGGGGCGGAG
ZmNHX7-G-F2 GCTGTGGTTGCACTGCTAAA
ZmNHX7-G-R2 TGCAATAACAACCCCACTCA
AtNHX8-G-F TTCCGTACACCGTCGTTCT
AtNHX8-G-R CCCCATCAATTAACGTGGTC
At-actin-F GCCAATCCGGTGCTGGTAACA
At-actin-R CATACCAGATCCAGTTCCTCCTCCC
荧光定量PCR引物序列 ZmNHX7-Q-F TGGGTTGGACTTGAAAGAGG
RT-PCR primer sequences ZmNHX7-Q-R AACACAATGCCACCAGTGAA
GAPDH-F AGGATATCAAGAAAGCTATTAAGGC
GAPDH-R GTAGCCCCACTCGTTGTCG
基因亚细胞定位 ZmNHX7-L-F AGCAGGCTTTGACTTTATGGGCGGCGAGGCTGAGCCTGACA
Subcellular localization of gene ZmNHX7-L-R TGGGTCTAGAGACTTTCTACTGCTCCTGGGGCGGAGGCACG

表2

玉米NHX基因信息"

基因名称
Gene name		 基因序列号
Gene ID		 CDS序列长度
CDS sequence length (bp)
ZmNHX1 GRMZM2G037342 1641
ZmNHX2 GRMZM2G063492 1641
ZmNHX3 GRMZM2G118019 1593
ZmNHX4 GRMZM2G027851 1620
ZmNHX5 GRMZM2G090149 1620
ZmNHX6 GRMZM2G067747 1611
ZmNHX7 GRMZM2G098494 3411

图1

拟南芥、玉米NHX家族进化树(A)及ZmNHX7分支的蛋白结构(B)"

图2

荧光定量PCR分析ZmNHX7基因在玉米不同组织中的表达 V1: 玉米第1片叶完全展开; V7: 玉米第7片叶完全展开; R2: 籽粒建成。误差线表示3次生物学重复的标准差, 以基因V1时期根的相对表达水平为对照。"

图3

ZmNHX7基因的诱导表达情况 A: ZmNHX7基因在不同浓度LiCl处理5 h后的表达量分析; B: ZmNHX7基因在不同浓度NaCl处理5 h后的表达量分析。误差线代表3个独立重复的标准差, *和**分别表示不同浓度离子胁迫条件下ZmNHX7基因相对表达量与对照在P < 0.05和P < 0.01水平差异显著。"

图4

ZmNHX7的亚细胞定位 A: YFP空载在烟草叶片中的表达; B: ZmNHX7-YFP在烟草叶片中的表达; C: 加上marker的ZmNHX7-YFP在烟草叶片中的表达。"

图5

通过PCR鉴定ZmNHX7基因在拟南芥中的转入与表达 A: RT-PCR检测AtNHX8基因在WT和nhx8中的转录表达; B: ZmNHX7基因在不同拟南芥株系中的表达; C: ZmNHX7基因在不同拟南芥株系中的转录表达。M: D2000 plus DNA marker。"

图6

拟南芥不同株系表型图及鲜重情况 A: WT、nhx8和COM1~COM4在含有0、5、15 mmol L-1 LiCl, 75 mmol L-1 NaCl的MS培养基上的生长表型图; B: WT、nhx8和COM1~COM4在含有离子胁迫的培养基上生长7 d后的鲜重情况(数据显示为平均值±SD, n = 20。图柱上不同大写字母表示在0.01水平上同一处理下的材料间差异显著)。"

图7

拟南芥根长及其数据统计 A: WT、nhx8、COM1和COM4在含有0、5、10 mmol L-1 LiCl, 50、100 mmol L-1 NaCl的MS培养基上的根长表型图; B: WT、nhx8、COM1、COM4在含有离子胁迫的培养基上生长7 d后的根长数据统计(数据显示为平均值±SD, n = 10。图柱上不同大写字母表示在0.01水平上同一处理下的材料间差异显著)。"

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