Cloning and expression study of a high-affinity nitrate transporter gene from Zea mays L

Abstract

A nitrate transporter gene, named B46NRT2.1, from salt-tolerant Zea mays L. B46 has been cloned. B46NRT2.1 contained the same domain belonging to the major facilitator superfamily (PLN00028). The results of the phylogenetic tree indicated that B46NRT2.1 exhibits sequence similarity and the closest relationship with those known nitrate transporters of the NRT2 family. Through RT-qPCR, we found that the expression of B46NRT2.1 mainly happens in the root and leaf. Moreover, the treatment with NaCl, Na₂CO₃, and NaHCO₃ could significantly increase the expression of B46NRT2.1. B46NRT2.1 was located in the plasma membrane. Through the study of yeast and plant salt response brought by B46NRT2.1 overexpression, we have preliminary knowledge that the expression of B46NRT2.1 makes yeast and plants respond to salt shock. There are 10 different kinds of cis-acting regulatory elements (CRES) in the promotor sequences of B46NRT2.1 gene using the PlantCARE web server to analyze. It mainly includes hormone response, abscisic acid, salicylic acid, gibberellin, methyl jasmonate, and auxin. The B46NRT2.1 gene's co-expression network showed that it was co-expressed with a number of other genes in several biological pathways, including regulation of NO₃ long-distance transit, modulation of nitrate sensing and metabolism, nitrate assimilation, and transduction of Jasmonic acid-independent wound signal. The results of this work should serve as a good scientific foundation for further research on the functions of the NRT2 gene family in plants (inbred line B46), and this research adds to our understanding of the molecular mechanisms under salt tolerance.

Keywords: High-affinity nitrate transporter gene; Zea mays; salt shock.

Figure 1.

The analysis of conserved domain of B46NRT2.1. The gene B46NRT2.1 mainly contains specific hits (PLN00028, MFS_NRT2_like, MFS_2), nonspecific hits (2A0108, NarK), and superfamilies (MFS superfamily, PLN00028, 2A0108 superfamily, NarK superfamilies).

Figure 2.

Phylogenetic tree of the B46NRT2.1 using MEGA7 software. The bar represents 0.1 aa substitution for pre site. (Sv: Setaria viridis; Si: Setaria italica; Pm: Panicum miliaceum; Ph: Panicum hallii; Os: Oryza sativa; Sb: Sorghum bicolor Me: Manihot esculenta).

Figure 3.

The alignment of the B46NRT2.1 amino acid sequence with NRT2.1 proteins from other species. (Sv: Setaria viridis; Si: Setaria italica; Pm: Panicum miliaceum; Ph: Panicum hallii; Os: Oryza sativa; Sb: Sorghum bicolor Me: Manihot esculenta;)

Figure 4.

The expression level of the B46NRT2.1 in different organs were examined using RT-qPCR. cDNA was obtained from different organs (Roots, Shoots, Leaves, Seeds) of Zea mays L.B46 lines, and the transcript abundances was detected by RT-qPCR to investigate B46NRT2.1 expression in the different organs of Zea mays L.B46 lines. ***Significance P < .001, ****Significance P < .0001.

Figure 5.

RT-qPCR analysis of B46NRT1.1 expression level of Zea mays L. under different salt shock. B46 lines were treated with 300 mM NaCl (a), 100 mM Na₂CO₃ (b), or 150 mM NaHCO₃ (c) for 0 h, 6h, 12 h, 24 h, 36 h, and 72 h respectively, then cDNA was obtained from Zea mays L. with different salt treatment, and the transcript abundances was detected by RT-qPCR to investigate the impact of salinity on B46NRT2.1 expression.

Figure 6.

Subcellular localization of the B46NRT2.1. Imaging was captured respectively visualized under bright channel (Bright), green fluorescent protein channel (GFP), and merged GFP and bright light channel (GFP + Bright) in N. benthamiana cells. A. Expression of the GFP protein in N. benthamiana. B. Expression of the B46NRT2.1-GFP fusion protein in N. benthamiana.

Figure 7.

The growth rate of E. coli cells with the expression of B46NRT2.1 or not under different salt treatment. The protein expression strain BL21 was transformed by PGEX vector or pGEX-B46NRT2.1 plasmid. These E. coli cells were induced by 1 mM IPTG for 1,2,3,4,5 h under different salt treatment, and then the growth rate of them were measured using spectrometer (OD₆₀₀). A. No salt treatment (CK). B. 0.8 M NaCl treatment. C. 0.1 M Na₂CO₃ treatment. D. 0.2 M NaHCO₃ treatment.

Figure 8.

The growth of yeast cells carrying the pYES2 or pYES2-B46NRT2.1 under salt shock. Ten-fold dilutions of yeast cells containing pYES2 (upper line) and pYES2-B46NRT2.1 (lower line) were spotted onto solid YPG media supplemented with the 30 mM NaHCO₃, 1 M NaCl, and 20 mM Na₂CO₃ treatment. No treatment was used as a control with YPD media.

Figure 9.

Identification of B46NRT2.1 overexpression A. thaliana lines. The expression contrasting of B46NRT2.1 in the wild-type and overexpression lines were measured using RT-qPCR. WT: Wide type. OTL1-6: B46NRT2.1 transgenic A. thaliana lines.

Figure 10.

Seed germination in A. thaliana wild-type and B46NRT2.1 overexpression plants under different stresses. Seed of T3 overexpression A. thaliana and wild-type were grown on 1/2 MS medium supplemented with 0 mM (CK); 100 mM NaCl, 125 mM NaCl, 150 mM NaCl; 3 mM Na₂CO₃, 5 mM Na₂CO₃, 7 mM Na₂CO₃; 3 mM NaHCO₃, 5 mM NaHCO₃, or 7 mM NaHCO₃, respectively. WT: Wild-type A. thaliana. #1, #2, #3 and #4: B46NRT2.1 overexpression A. thaliana lines.

Figure 11.

The phenotype of the WT and overexpression Arabidopsis lines seedlings under different stresses. The sterilized Arabidopsis seedlings were evenly plated on 1/2 MS with 200 mM NaCl, 20 mM Na₂CO₃, 20 mM NaHCO_3, respectively. No treatment (CK) was used as a control. WT: Wild-type A. thaliana. #1, #2 and #3: B46NRT2.1 overexpression lines.

Figure 12.

Predicted cis elements in the promoter of the B46NRT2.1 gene. The 2000 bp region upstream of the initiation codon (ATG) of the NRT2.1 gene was analyzed to examine the promoter sequences by the PlantCARE online server.

Figure 13.

Co-expression network analyses of B46NRT2.1. Co-expression network analyses of B46NRT2.1 using the TomExpress platforms.