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Acta Agronomica Sinica ›› 2020, Vol. 46 ›› Issue (8): 1185-1194.doi: 10.3724/SP.J.1006.2020.93062

• CROP GENETICS & BREEDING · GERMPLASM RESOURCES · MOLECULAR GENETICS • Previous Articles     Next Articles

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 Online:2020-08-12 Published:2020-04-03
  • Contact: Jun ZHENG E-mail:zhengjun02@caas.cn
  • Supported by:
    National Key Research and Development Program of China(2016YFD0101002)

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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

Table 1

Primer sequences"

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

Table 2

Maize NHX gene information"

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

Fig. 1

Phylogenetic tree of NHX gene family in Arabidopsis thaliana and maize (A) and ZmNHX7 branched protein structure (B)"

Fig. 2

Expression analysis of ZmNHX7 gene in different tissues of maize by real-time fluorescence quantitative PCR V1: the first leaf of maize was fully expanded; V7: the seventh leaf of maize was fully expanded; R2: grain formation period. The error bar shows the standard deviation of three biological replicates, the relative expression level in root (V1) was used as the control."

Fig. 3

Induced expression of ZmNHX7 gene A: analysis of the expression level of ZmNHX7 after treatment with different concentrations of LiCl for 5 hours; B: analysis of the expression level of ZmNHX7 after treatment with different concentrations of NaCl for 5 hours. error bars represent standard errors of three independent repetitions; * and **: the relative expression of ZmNHX7 under different concentrations of ion stress is significantly different from the content at the 0.05 and 0.01 probability levels, respectively."

Fig. 4

Subcellular localization of ZmNHX7 A: localization of YFP fluorescence in tobacco leaves; B: localization of ZmNHX7-YFP fluorescence in tobacco leaves; C: localization of ZmNHX7-YFP and marker fluorescence in tobacco leaves."

Fig. 5

Identification of the transfer and expression of ZmNHX7 by PCR A: RT-PCR was used to verify the transcription of AtNHX8 gene in WT and nhx8; B: expression of ZmNHX7 gene in different Arabidopsis lines; C: transcriptional expression of ZmNHX7 gene in different Arabidopsis lines. M: D2000 plus DNA marker."

Fig. 6

Phenotype and fresh weight of different Arabidopsis strains A: germination and subsequent growth of wild type, nhx8 and COM1-COM4 plants under MS agar medium and MS medium containing 5 or 15 mmol L-1 LiCl, or 75 mmol L-1 NaCl. B: the fresh weight of wild type, nhx8 and COM1-COM4 after seven days of growth on medium containing ionic stress (the data are shown as mean ± SD, n = 20. Bars superscripted by different capitals are significantly different at P < 0.01 within a treatment)."

Fig. 7

Arabidopsis root length and data statistics A: root length phenotype of WT, nhx8, COM1, and COM4 on MS medium containing 0, 5, 10 mmol L-1 LiCl, and 50, 100 mmol L-1 NaCl. B: the root length of WT, nhx8, COM1, and COM4 after seven days of growth on medium containing ionic stress (The data are shown as mean ± SD, n = 10. Bars superscripted by different capitals are significantly different at the P < 0.01 within a treatment)."

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