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Acta Agronomica Sinica ›› 2023, Vol. 49 ›› Issue (11): 2966-2977.doi: 10.3724/SP.J.1006.2023.21063

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

Gene expression characteristics of TaNRT/TaNPF family in wheat cultivars with different nitrogen efficiency

WANG Lu-Lu1(), YI Zi-Bo1, WANG Hao-Zhe1, NAI Fu-Rong2, MA Xin-Ming1, ZHANG Zhi-Yong1,*(), WANG Xiao-Chun1,2,*()   

  1. 1Co-constuction State Key Laboratory of Wheat and Maize Crop Science, Henan Agricultural University, Zhengzhou 450002, Henan, China
    2Department of Biochemistry, College of Life Science, Henan Agricultural University, Zhengzhou 450002, Henan, China
  • Received:2022-09-13 Accepted:2023-04-17 Online:2023-11-12 Published:2023-05-16
  • Contact: 王小纯, E-mail: xiaochun.w@163.com; 张志勇, E-mail: zhiyongzhang@henau.edu.cn
  • Supported by:
    National Natural Science Foundation of China(32071956)

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Abstract:

Nitrogen is one of the essential elements for wheat growth and development, and NO3--N is the main form of nitrogen that wheat obtains from soil. NRT/NPF genes family encode membrane transporters, which are mainly involved in NO3--N absorption, transport, and allocation in plants. In order to understand the relationship between NRT/NPF family and nitrogen utilization in wheat, the relative expression characteristic of TaNRT/TaNPF family in flag leaves of N-efficient wheat cultivars Zhoumai 27 (ZM27) and N-inefficient wheat cultivars Aikang 58 (AK58) at flowering stage were studied with the second-generation sequencing technology. The results showed that 386 genes of TaNRT/TaNPF family were identified in the second generation transcriptome database. Compared with AK58, there were 27, 16, and 23 differentially expressed genes in ZM27 in reducing (N120), normal (N225), and excessive (N330) nitrogen treatments. There were 16 (59.26%), 12 (75%), and 19 (82.61%) up-regulated genes in ZM27, respectively. Seven genes were down-regulated in ZM27 in reducing nitrogen treatment. The relative expression level of TaNPF8.1 was the highest and significantly up-regulated by 1.5 times in nitrogen excessive condition. In conclusion, the relative expression of TaNRT/TaNPF family genes was regulated by nitrogen application rate and cultivar. Wheat network database showed that the relative expression of TaNRT/TaNPF family had tissue specificity and chromosomal preference. The highest expression level of TaNPF8.1 in flag leaf was located on chromosome 3A, and the root specific expression TaNRT2.2 and TaNRT3.1 were mainly distributed on chromosome 6. The stem specific expression of TaNPF4.5 was mainly distributed on chromosome 2. The qRT-PCR of TaNRT/TaNPF genes were consistent with the results of the second-generation transcriptome and network data. Interaction analysis of TaNPF8.1, TaNPF4.5, and TaNRT3.1 revealed that NO3--N transport may also require the collaborative participation of transcription factor MYB, chlorophyll A-B binding protein, and chaperone protein. These findings laid a foundation for further studies on the relationship between TaNRT/TaNPF family expression and nitrogen uptake and utilization.

Key words: wheat, NRT/NPF, nitrogen use efficiency, different expression, tissue specificity

Table 1

qRT-PCR primer sequences"

基因名称
Gene name		 正向引物序列
Forward sequence (5′-3′)		 反向引物序列
Reverse sequence (5′-3′)
NRT2.2 CCTTGGTATCATCTCCGGGC GAGGGTGACAGGAAGAGTGC
NRT3.1 CCCCCAGCTCTTCTCTTGC TCACCGGCAGCTTGGAGA
NPF4.5 AGCAGCAACCTACAAGCAGT TGGAAGCTCTCACTCCTCCA
NPF5.102D GGAAACCACGCACAGAAGC CGTGGGTGTTTTGTTCCTGTC
NPF5.101A CCCTCGTCCTGTAGCTTGAC CCAGCAGAGGACGCCATTAA
NPF8.13A TCAGAGAACGCAACCACTGT CCTGCAGGCCTTTTGATTCG
NPF8.34A ATTCCTTCTCCCTCCCCGAA GCTGGGATACTCTGCTCGGA
NPF8.34D CCCTTCCCCTTGCTTGCTTA TCGGGGTGGGAGTTGAGTTA
NPF8.31B AACGTGAAAGGGGGAGGGAT GGTTTGCCGTTGTGATTTGC
CHS2 CACCACAATAATCCTTGCTA ACTTATGACCCAATTACTGAAT
PTR1 AAGCGGGCTGATGGTGATGG CGGCGGTGACCAGGTAGAAC
POD70 CTGAGGCGAACAGCGACCTC CGGGCTCAGCTGCTTCTTGT
RPP13 TGGGTTCGGTGGCATAGGGA CCCAAGCCCGTGCTTCGTAT
ATPase CGAGGCCACCAATGACG AGTATGGTTTCAAGAAGGCGTC
TEF TCGTGGTCATTGGCCACG CAGCACAGTCAGCCTGGGAG

Fig. 1

Differentially expression genes of TaNRT/TaNPF family A: data are from the online database (https://www.wheatproteome.org/); blue-white-red indicates the relative expression level from low to high, B: data are from the second-generation transcriptome sequencing."

Fig. 2

Differential expression characteristics of TaNRT/TaNPF family in flag leaves of different cultivars with different nitrogen levels A: the number of TaNRT/TaNPF DEGs. red arrow: the up-regulated gene; green arrow: down-regulated gene. B: the number of up-regulated genes in TaNRT/TaNPF family in different nitrogen levels; C: the number of down-regulated genes in TaNRT/TaNPF family in different nitrogen levels."

Table S1

Analysis of differential gene expression in wheat cultivars with different nitrogen efficiency"

类型
Type		 基因名称
Gene name		 AK58 N120 AK58 N225 AK58 N330 ZM27 N120 ZM27 N225 ZM27 N330
Up-regulated DEGs NPF2.3 TraesCS4A03G1098900 0.14 0.29 0.03 0.69 0.35 0.50
NPF2.11 TraesCS5A03G0008900 2.79 3.03 2.19 5.70 5.64 5.08
TraesCS5B03G0001600 2.39 2.12 2.06 5.86 6.84 5.24
TraesCS5B03G0102800 8.11 6.37 6.52 11.29 11.94 9.35
NPF4.4 TraesCS4A03G0599700 0.28 0.41 0.36 0.73 0.75 1.01
NPF5.1 TraesCS7A03G1119900 2.33 2.78 2.50 3.05 3.92 3.64
TraesCS7D03G1067400 0.50 0.82 0.56 0.64 0.79 1.05
NPF5.8 TraesCS7B03G0265800 0.28 0.39 0.27 0.56 0.50 0.56
TraesCS7D03G0440800 0.60 1.17 0.91 1.28 1.37 1.45
NPF5.10 TraesCS3A03G0903800 0.59 0.75 0.43 0.71 1.01 0.93
NPF5.13 TraesCS2D03G1323700 0.88 1.19 1.23 1.11 1.58 1.93
NPF6.2 TraesCS1B03G0077900 0.33 0.39 0.37 0.19 0.23 0.95
NPF7.3 TraesCS6D03G0593500 0.21 0.29 0.16 0.44 0.37 0.40
NPF8.1 TraesCS3A03G0922900 35.44 37.45 34.96 51.56 52.52 53.18
TraesCS3A03G0923200 3.28 3.04 3.17 5.67 6.21 5.36
TraesCS3D03G0852600 0.82 0.77 0.62 0.72 0.63 0.96
TriticumnewGene_8087 17.36 16.63 15.86 25.28 25.27 24.94
NPF8.3 TraesCS7A03G1290100 0.09 0.13 0.58 1.02 0.54 1.13
TraesCS7B03G0835300 1.98 1.99 1.65 2.60 2.77 2.73
Down-regulated DEGs NRT2.4 TraesCS7B03G0882400 5.31 4.37 4.39 0.19 0.09 0.25
NPF2.11 TraesCS5D03G0010900 2.53 1.53 1.67 1.49 1.42 1.41
NPF4.6 TraesCS5D03G0163200 2.17 1.20 1.82 1.29 0.50 0.44
NPF5.2 TraesCS4D03G0779400 1.79 1.32 1.56 1.07 0.98 1.37
NPF5.10 TraesCS2A03G1350700 1.89 2.30 2.51 0.99 1.33 1.26
TraesCS2B03G1540700 1.55 1.39 1.80 0.94 1.09 1.21
TraesCS3A03G0902900 0.93 0.83 0.89 0.52 0.72 0.64
NPF8.3 TraesCS6B03G1146900 1.10 0.98 1.38 0.54 0.79 0.92
TraesCS7B03G0839000 1.56 1.53 2.33 0.24 0.43 0.56
TraesCS7B03G0839200 7.12 7.14 6.78 4.81 5.00 4.94
NPF8.5 TraesCS2D03G0016800 2.02 1.59 1.79 1.08 1.52 1.51

Fig. 3

Validation of transcriptome data by qRT-PCR A: the relative expression level of selected differentially expressed genes detected by qRT-PCR; B: FPKM values corresponding to selected genes. AK58: Aikang 58; ZM27: Zhoumai 27; N120: nitrogen reduction 120 kg hm-2; N225: normal nitrogen 225 kg hm-2; N330: excess nitrogen 330 kg hm?2; NPF5.10-1A, NPF5.10-2D, NPF8.1-3A, NPF8.3-1B, NPF8.3-4A, NPF8.3-4D: nitric acid transporter gene; CHS2: Chalcone synthase2-like; PTR1: Peptide transporter1; POD70: Peroxidase70; RPP13: disease resistance protein RPP13."

Fig. 4

Effects of cultivars and nitrogen levels on the relative expression level of TaNRT/TaNPF family genes in different tissues of wheat Blue-white-red indicates gene relative expression level from low to high. TaNPF8.1, TaNPF4.5, TaNRT2.2, TaNRT3.1: nitric acid transporter. Abbreviations are the same as those given in Fig. 3."

Fig. 5

Functional prediction of differentially expressed genes in TaNRT/TaNPF families Blue-white-red indicates gene relative expression level from low to high."

Fig. 6

Distribution of TaNRT/TaNPF genes family on wheat chromosomes A: the distribution of tissue-specific TaNRT/TaNPF gene chromosomes; B: the distribution of TaNRT/TaNPF gene regulated by nitrogen chromosomes."

Fig. 7

Protein-protein interaction (PPI) network of tissue-specific TaNRT/TaNPF Proteins are represented by nodes and interactions by wires. Gray: transcription factors; Yellow: inoraganicion transport and metabolism; Prasinous: aspartic acid protease; Red: light-inducible protein; Purple: Bowman-Birk type proteinase inhibitor; Green: crocetin glucosyltransferase; Black: GPI-anchored protein; Blue: cytochrome; Sapphire: chaperone protein; Rosy: MAH1; Buff: NAD kinase; Reseda: nitric acid transporter; Indipink: Cyanidin 3-O-rutinoside 5-O-glucosyltransferase."

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