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Acta Agronomica Sinica ›› 2019, Vol. 45 ›› Issue (3): 365-380.doi: 10.3724/SP.J.1006.2019.84099

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

Bioinformatics analysis and response to nitrate-cadmium stress of NRT1.5 and NRT1.8 family genes in Brassica napus

Gui-Hong LIANG1,2,Ying-Peng HUA1,2,Ting ZHOU1,2,Qiong LIAO1,2,Hai-Xing SONG1,2,Zhen-Hua ZHANG1,2,*()   

  1. 1 College of Resource and Environment, Hunan Agricultural University, Changsha 410128, Hunan, China
    2 Southern Regional Collaborative Innovation Center for Grain and Oil Crops in China, Changsha 410128, Hunan, China
  • Received:2018-07-18 Accepted:2018-10-08 Online:2019-03-12 Published:2018-11-02
  • Contact: Zhen-Hua ZHANG E-mail:zhzh1468@163.com
  • Supported by:
    This study was supported by the National Key R&D Program of China(2017YFD0200100);This study was supported by the National Key R&D Program of China(2017YFD0200103);the China Agriculture Research System

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Abstract:

The absorption and transportation of nitrate in plants require the assistance of nitrate transporters (NRTs). The expression of two members of the NRT1 family, including NRT1.5 and NRT1.8 genes was strongly induced by nitrate, and regulated the long-distance transport and distribution of nitrate between roots and shoots in Arabidopsis. NRT1.5 and NRT1.8 homologous genes in B. rapa, B. oleracea, and B. napus were identified by bioinformatics with the basic sequences of AtNRT1.5 and AtNRT1.8 and analyzed in gene structures and proteins molecular characterization, gene copy number variations, chromosome locations, evolutionary relationship tree, proteins conservative sequence alignment and the transmembrane domains. NRT1.5 and NRT1.8 responsive to the low concentration nitrate and cadmium stress were also determined by transcriptome analysis and co-expression network analysis, showing that NRT1.5 and NRT1.8 proteins belong to major facilitator superfamily (MFS) and peptide transporter (PTR) with the conservative transmembrane domains and motifs (F-Y-L-A-L-N-L-G-S-L) in B. rapa, B. oleracea, and B. napus. High-throughput transcriptome analysis showed that the expression of NRT1.5 gene was up-regulated and the NRT1.8 was down-regulated by low concentration nitrate treatment for 72 h in roots, which caused more nitrate transferred from roots to shoots. On the contrary, the ethylene/jasmonic acid-NRT signaling module could promote NRT1.8 up-regulation and inhibit the expression of NRT1.5 by cadmium treatment. So that more nitrate transported from shoots to roots and improved the ability of plants to resist cadmium stress. This study is valuable for the research of biological functions of NRT1.5 and NRT1.8 family genes in B. napus and the responses to nitrate-cadmium stress. Our results also provide references for the bioinformatic study of NRT1.5 and NRT1.8 family genes in other plant species.

Key words: Brassica napus, bioinformatics, NRT1.5, NRT1.8, nitrate, cadmium

Table 1

Molecular characterization of NRT1.5 and NRT1.8 genes in B. rapa, B. oleracea, and B. napus"

基因名
Gene name		 基因编号
Gene ID		 分区
Block		 亚类
Subgenome		 物理位置
Physical position		 编码区长度
CDS (bp)		 外显子/内含子Exon/intron
NRT1.5
BraA5.NRT1.5 Bra010165 B MF2 15109908-15113350 1815 5/4
BraA9.NRT1.5 Bra023267 B LF 20166714-20170510 1833 5/4
BolC5.NRT1.5a Bol020904 B MF2 13227461-13230901 1812 5/4
BolC5.NRT1.5b Bol022226 B LF 10183303-10186752 1236 5/4
BnaA5.NRT1.5 BnaA05g35790D B MF2 1269482-1274007 1845 6/5
BnaA9.NRT1.5 BnaA09g24330D B LF 17085147-17089456 1863 6/5
BnaC5.NRT1.5a BnaC05g28620D B MF2 26898087-26903213 1845 6/5
BnaC5.NRT1.5b BnaC05g24580D B LF 19037761-19041900 1860 6/5
NRT1.8
BraA1.NRT1.8 Bra013547 U LF 6243271-6245298 1752 4/3
BraA3.NRT1.8 Bra038763 U MF1 24230588-24232594 1752 4/3
BolC1.NRT1.8 Bol028440 U LF 8086050-8088075 1752 4/3
BolC6.NRT1.8 Bol024295 U MF1 42081659-42082729 897 3/2
BnaA1.NRT1.8 BnaA01g11510D U LF 5715790-5718126 1752 4/3
BnaA3.NRT1.8 BnaA03g44820D U MF1 22774854-22776862 1752 4/3
BnaC7.NRT1.8 BnaC07g36810D U MF1 38737319-38739325 1752 4/3
BnaCn.NRT1.8 BnaCnng78690D U LF 80520183-80521816 1167 3/2

Table 2

Molecular characterization of NRT1.5 and NRT1.8 proteins in B. rapa, B. oleracea, and B. napus"

基因名
Gene name		 氨基酸数Amino acids 主要氨基酸 Major amino acids 碱性氨基酸Arg+Lys 酸性氨基酸Asp+Glu 分子量MW(kD) 等电点pI 不稳定系数Instability index 亲水性GRAVY 脂肪指数Aliphatic index
NRT1.5
BraA5.NRT1.5 604 Leu, Ser 58 63 67.64 5.93 34.13 0.096 88.79
BraA9.NRT1.5 610 Leu, Ser 59 62 68.17 6.29 31.20 0.073 89.18
BolC5.NRT1.5a 603 Leu, Ser 58 63 67.45 5.93 33.33 0.094 88.77
BolC5.NRT1.5b 411 Leu, Ser 38 44 45.80 5.63 27.54 -0.006 86.13
BnaA5.NRT1.5 614 Leu, Ser 59 64 68.72 5.93 34.82 0.094 88.62
BnaA9.NRT1.5 620 Leu, Ser 60 64 69.41 6.14 32.08 0.082 89.47
BnaC5.NRT1.5a 614 Leu, Ser 59 64 68.71 5.93 34.68 0.094 88.45
BnaC5.NRT1.5b 619 Leu, Ser 59 62 69.35 6.35 33.07 0.094 89.77
NRT1.8
BraA1.NRT1.8 583 Leu, Ser 48 51 64.72 6.22 27.23 0.209 91.66
BraA3.NRT1.8 583 Leu, Ala 51 51 64.55 7.06 29.40 0.218 94.70
BolC1.NRT1.8 583 Leu, Ser 48 52 64.67 6.05 28.13 0.211 92.16
BolC6.NRT1.8 298 Leu, Ala 26 24 32.84 8.18 18.64 0.088 88.36
BnaA1.NRT1.8 583 Leu, Ser 48 51 64.60 6.22 27.84 0.211 91.17
BnaA3.NRT1.8 583 Leu, Ala 52 51 64.60 7.52 27.69 0.208 94.70
BnaC7.NRT1.8 583 Leu, Ser 53 50 64.67 8.18 27.88 0.207 94.85
BnaCn.NRT1.8 388 Leu, Ser 37 38 43.81 6.60 32.07 0.110 87.96

Fig. 1

Copy number variations of NRT1.5 and NRT1.8 genes in B. rapa, B. oleracea, B. napus, and A. thaliana The number at the top of the histogram is the number of genes copied for that species."

Fig. 10

Supplementary fig. 1 Chromosomal localization of NRT1.5 and NRT1.8 genes in B. rapa, B. oleracea, B. napus, and A. thaliana. Fig. a shows the chromosomal localization of NRT1.5 genes in A. thaliana, B. napus, B. oleracea, and B. rapa. Fig. b shows the chromosomal localization of NRT1.8 genes in A. thaliana, B. napus, B. oleracea, and B. rapa."

Fig. 2

Gene structure characteristics of NRT1.5 and NRT1.8 in B. rapa, B. oleracea, B. napus, and A. thaliana. Fig. a and Fig. b show the gene structure characteristics of NRT1.5 and NRT1.8 respectively in A. thaliana, B. rapa, B. oleracea, and B. napus. The yellow parts represent the CDS sequence and the blue parts represent the upstream and downstream genes. The black lines represent the intron of genes."

Fig. 3

Phylogenetic relationships of the NRT1.5 and NRT1.8 proteins in diverse species. Fig.a and Fig.b show the phylogenetic relationships of NRT1.5 and NRT1.8 proteins respectively in dicots and monocots. The green parts represent the dicots, including Arabidopsis thaliana, Brassica rapa, Brassica oleracea, Brassica napus, and Populus euphratica. The red parts represent the monocots, including Sorghum bicolor, Oryza sativa, Setaria italica, Zea mays, and Brachypodium distachyon."

Fig. 4

Synonymous nucleotide substitution rates (Ks) and non-synonymous nucleotide substitution rates (Ka) of NRT1.5 and NRT1.8 proteins in B. rapa, B. oleracea, and B. napus Fig. a, Fig. b, and Fig. c show synonymous nucleotide substitution rates and non-synonymous nucleotide substitution rates of NRT1.5 proteins in B. rapa, B. oleracea, and B. napus, respectively. Fig. d, Fig. e, and Fig. f show synonymous nucleotide substitution rates and non-synonymous nucleotide substitution rates of the NRT1.8 proteins in B. rapa, B. oleracea, and B. napus, respectively."

Fig. 5

Conservative sequence alignment of NRT1.5 and NRT1.8 proteins in B. rapa, B. oleracea, B. napus, and A. thaliana. Fig. a and Fig. b show the conservative sequence alignment of NRT1.5 and NRT1.8 proteins respectively in A. thaliana, B. rapa, B. oleracea, and B. napus. The blue parts show the conservative sequence (F-Y-L-A-L-N-L-G-S-L) of peptide transporter."

Fig. 6

Characterization of conserved motifs in the NRT1.5 and NRT1.8 proteins in B. rapa, B. oleracea, B. napus, and A. thaliana. Fig. a and Fig. d show the phylogenetic relationships of NRT1.5 and NRT1.8 proteins respectively in A. thaliana, B. rapa, B. oleracea, and B. napus. Fig. b and Fig. e show the motives of NRT1.5 and NRT1.8 proteins respectively in A. thaliana, B. rapa, B. oleracea, and B. napus. Fig. c and Fig. f show the sequence of conserved motif of NRT1.5 and NRT1.8 proteins."

Fig. 7

Transmembrane domains of NRT1.5 and NRT1.8 proteins in B. napus Fig. a to Fig. d show four transmembrane domains of NRT1.5 protein in B. napus. Fig. a, Fig. b, Fig. c, and Fig. d show the protein of Bna.A5.NRT1.5, Bna.A9.NRT1.5, Bna.C5.NRT1.5a, and Bna.C5.NRT1.5b, respectively. Fig. e to Fig. h show four transmembrane domains of NRT1.8 protein in B. napus. Fig. e, Fig.f, Fig. g, and Fig. h show the protein of Bna.A1.NRT1.8, Bna.A3.NRT1.8, Bna.C7.NRT1.8, and Bna.Cn.NRT1.8, respectively."

Fig. 8

Response to low concentration nitrate and co-expression network analysis of NRT1.5 and NRT1.8 genes in B. napus Fig. a shows the gene expression abundance of NRT1.5 family genes in shoots and roots of B. napus with low nitrate treatment for 0 h, 3 h, 72 h. Fig. b shows the co-expression network analysis of NRT1.5 family genes with low nitrate treatment. Fig. c shows the gene expression abundance of NRT1.8 family genes in shoots and roots of B. napus with low nitrate treatment for 0 h, 3 h, 72 h. Fig. d shows the co-expression network analysis of NRT1.8 family genes with low nitrate treatment. FPKM means the fragments per kilobase of exon model per million mapped reads."

Fig. 9

Response to cadmium stress and co-expression network analysis of NRT1.5 and NRT1.8 genes Fig. a and Fig. b show the gene expression abundance of NRT1.5 family genes in shoots and roots of B. napus with cadmium stress. Fig. c shows the co-expression network analysis of NRT1.5 family genes with cadmium stress. Fig. d and Fig. e show the gene expression abundance of NRT1.8 family genes in shoots and roots of B. napus with cadmium stress. Fig. f shows the co-expression network analysis of NRT1.8 family genes with cadmium stress. FPKM means the fragments per kilobase of exon model per million mapped reads. The significant difference shown in the figure is the pairwise comparison between control and cadmium treatment, and the figure without significant difference is not marked. Data are shown as means±SE (n = 3) and the different lowercases above text are used to show statistical significance (Ρ < 0.05)."

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