Scientia Agricultura Sinica ›› 2023, Vol. 56 ›› Issue (19): 3723-3746.doi: 10.3864/j.issn.0578-1752.2023.19.003

• SPECIAL FOCUS: GENE FUNCTION AND BREEDING IN COTTON • Previous Articles     Next Articles

Genome-Wide Identification and Expression Analysis of NLP (NIN- Like Protein) Transcription Factor Gene Family in Cotton

DING GuoHua(), XIAO GuangHui(), ZHU LiPing()   

  1. College of Life Sciences, Shaanxi Normal University, Xi’an 710119
  • Received:2022-11-07 Accepted:2023-01-18 Online:2023-10-01 Published:2023-10-08
  • Contact: XIAO GuangHui, ZHU LiPing

Abstract:

【Objective】To explore the structure and evolution characteristics of cotton NLP transcription factors in the whole genome, and further understand their expressions patterns, so as to lay a foundation for the further function research and utilization of NLP genes. 【Method】The NLP transcription factor family members in the whole genomes of four cotton species, Gossypium arboreum (G. arboreum, Ga), Gossypium raimondii (G. raimondii, Gr), Gossypium barbadense (G. barbadense, Gb) and Gossypium hirsutum (G. hirsutum, GH), were identified using two strategies, BLASTP and HMM search. Further bioinformatics analysis was carried out on the confirmed cotton NLP family members. The molecular weights, theoretical isoelectric points and other physical and chemical properties were predicted using online software Expasy; the MEGA 7 software was used to build the phylogenetic tree; protein conservative motifs were analyzed through MEME website; online software GSDS 2.0 was used to analyze gene structures; TBtools was used to view the chromosome localizations; McscanX was used to analyze the replication genes of cotton NLP family members; the PlantCARE website was used to predict the cis-acting elements in the promoters of cotton NLP family genes. The heat maps of cotton NLP genes expression levels of different tissues and under abiotic stresses were drawn through TBtools to analyze the tissue expression characteristics and abiotic stresses response characteristics. The expressions of GHNLPs in cotton under nitrogen starvation and nitrogen resupply treatments were analyzed by RT-qPCR. 【Result】A total of 11, 11, 21 and 22 NLP members were screened from the four cotton protein databases of G. arboreum, G. raimondii, G. barbadense and G. hirsutum, respectively. These NLP family genes encoded 693-996 amino acids. The relative molecular masses ranged from 76.92-110.02 kDa and the theoretical isoelectric points were 5.13-7.77. The subcellular localization prediction results showed that almost all the NLP members located in the nucleus. Promoter analysis found a large number of cis-acting elements related to phytohormone and stress response. Phylogenetic analysis showed cotton NLPs were divided into three groups, I, II and III. Gene replication analysis showed that fragment replication was the main force for NLP members expansion in cotton. All the Ka/Ks values were less than 1, indicating that evolution of NLP family in cotton mainly underwent purification selection. The results of expression analysis also confirmed that GHNLPs responded to nitrogen starvation and nitrogen resupply. 【Conclusion】From the whole genome of G. arboreum, G. raimondii, G. barbadense, and G. hirsutum, 11, 11, 21 and 22 NLP transcription factor members were identified respectively. They had high conservatism and some degree of differences. The expression levels of GHNLPs changed significantly during nitrogen starvation and nitrogen resupply processes, which may play a role in the response of cotton to nitrate.

Key words: cotton, NLP transcription factor, nitrogen signal, bioinformatics

Table 1

Information of NLP gene family members in cotton"

基因
<BOLD>G</BOLD>ene
基因编号
<BOLD>G</BOLD>ene ID
长度
Lengh
(aa)
分子量
Mw
(kDa)
理论
等电点
pI
总平均亲水性
GRAVY
亚细胞定位
<BOLD>S</BOLD>ubcellular
localization
保守结构域
RWP-RK
(PF02042)a
保守结构域
PB1
(PF00564)b
GaNLP1-1 Cotton_A_33891 981 108.12 5.96 -0.382 细胞核Nucleus 1 1
GaNLP1-2 Cotton_A_02095 942 103.75 6.63 -0.399 细胞核Nucleus 1 1
GaNLP1-3 Cotton_A_00586 778 86.24 6.66 -0.358 细胞核Nucleus 1 1
GaNLP4-1 Cotton_A_12652 925 102.86 6.35 -0.452 细胞核Nucleus 1 1
GaNLP4-2 Cotton_A_04599 982 109.22 6.64 -0.429 细胞核Nucleus 1 1
GaNLP7-1 Cotton_A_08147 971 106.59 5.36 -0.358 细胞核Nucleus 1 1
GaNLP7-2 Cotton_A_33803 986 108.72 5.48 -0.341 细胞核Nucleus 1 1
GaNLP7-3 Cotton_A_27393 986 108.70 5.88 -0.399 细胞核Nucleus 1 1
GaNLP8-1 Cotton_A_04403 989 109.87 5.63 -0.450 细胞核Nucleus 1 1
GaNLP8-2 Cotton_A_29789 989 108.52 5.41 -0.421 细胞核Nucleus 1 1
GaNLP8-3 Cotton_A_16353 948 103.91 5.13 -0.386 细胞核Nucleus 1 1
GbNLP1A-1 Gbar_A01G017210.1 931 102.67 5.74 -0.419 细胞核Nucleus 1 1
GbNLP1A-2 Gbar_A12G003180.4 949 104.29 6.38 -0.413 细胞核Nucleus 1 1
GbNLP1A-3 Gbar_A03G022900.1 788 87.62 6.67 -0.357 细胞核Nucleus 1 1
GbNLP1D-1 Gbar_D01G018440.1 932 102.94 5.59 -0.425 细胞核Nucleus 1 1
GbNLP1D-2 Gbar_D12G004020.3 949 104.36 6.28 -0.423 细胞核Nucleus 1 1
GbNLP1D-3 Gbar_D02G024770.1 782 87.17 7.77 -0.389 细胞核Nucleus 1 1
GbNLP4A-1 Gbar_A01G009630.1 925 102.86 6.29 -0.446 细胞核Nucleus 1 1
GbNLP4A-2 Gbar_A05G017710.1 928 103.20 6.46 -0.511 细胞核Nucleus 1 1
GbNLP4D-1 Gbar_D01G010100.1 906 100.78 6.4 -0.464 细胞核Nucleus 1 1
GbNLP4D-2 Gbar_D05G018310.1 917 102.00 6.58 -0.521 细胞核Nucleus 1 1
GbNLP7A-1 Gbar_A09G019980.1 937 102.92 5.43 -0.361 细胞核Nucleus 1 1
GbNLP7A-3 Gbar_A02G010050.2 706 77.20 6.23 -0.371 细胞核Nucleus 1 1
GbNLP7D-1 Gbar_D09G019730.1 970 106.29 5.16 -0.356 细胞核Nucleus 1 1
GbNLP7D-2 Gbar_D08G012590.1 959 106.00 5.74 -0.357 细胞核Nucleus 1 1
GbNLP7D-3 Gbar_D03G009130.1 706 77.13 6.22 -0.357 细胞核Nucleus 1 1
GbNLP8A-1 Gbar_A02G000970.1 989 109.97 5.59 -0.462 细胞核Nucleus 1 1
GbNLP8A-2 Gbar_A02G009040.1 992 109.07 5.49 -0.412 细胞核Nucleus 1 1
GbNLP8A-3 Gbar_A10G015190.1 951 104.27 5.19 -0.397 细胞核Nucleus 1 1
GbNLP8D-1 Gbar_D02G001220.1 989 110.02 5.63 -0.475 细胞核Nucleus 1 1
GbNLP8D-2 Gbar_D02G011910.1 996 109.38 5.52 -0.408 细胞核Nucleus 1 1
GbNLP8D-3 Gbar_D10G012970.1 951 104.25 5.14 -0.396 细胞核Nucleus 1 1
GHNLP1A-1 GH_A01G1889 931 102.65 5.74 -0.419 细胞核Nucleus 1 1
GHNLP1A-2 GH_A12G0312 949 104.28 6.38 -0.419 细胞核Nucleus 1 1
GHNLP1A-3 GH_A03G2345 783 87.03 6.66 -0.365 细胞核Nucleus 1 1
GHNLP1D-1 GH_D01G1993 932 103.09 5.61 -0.428 细胞核Nucleus 1 1
GHNLP1D-2 GH_D12G0395 949 104.39 6.29 -0.434 细胞核Nucleus 1 1
GHNLP1D-3 GH_D02G2514 782 87.41 7.77 -0.405 细胞核Nucleus 1 1
GHNLP4A-1 GH_A01G1022 925 102.87 6.29 -0.446 细胞核Nucleus 1 1
GHNLP4A-2 GH_A05G1796 928 103.32 6.39 -0.515 细胞核Nucleus 1 1
GHNLP4D-1 GH_D01G1052 906 100.83 6.51 -0.478 细胞核Nucleus 1 1
GHNLP4D-2 GH_D05G1830 926 102.99 6.4 -0.508 细胞核Nucleus 1 1
GHNLP7A-1 GH_A09G2046 970 106.55 5.39 -0.360 细胞核Nucleus 1 1
GHNLP7A-2 GH_A08G1255 897 98.47 5.28 -0.330 细胞核Nucleus 1 1
GHNLP7A-3 GH_A02G1059 986 108.81 5.92 -0.408 细胞核Nucleus 1 1
GHNLP7D-1 GH_D09G1989 970 106.31 5.19 -0.357 细胞核Nucleus 1 1
GHNLP7D-2 GH_D08G1298 986 108.60 5.62 -0.338 细胞核Nucleus 1 1
GHNLP7D-3 GH_D03G0999 986 108.73 5.88 -0.396 细胞核Nucleus 1 1
GHNLP8A-1 GH_A02G0111 989 109.96 5.63 -0.467 细胞核Nucleus 1 1
GHNLP8A-2 GH_A02G0950 992 108.96 5.45 -0.421 细胞核Nucleus 1 1
GHNLP8A-3 GH_A10G1528 951 104.24 5.22 -0.397 细胞核Nucleus 1 1
GHNLP8D-1 GH_D02G0117 989 109.96 5.64 -0.467 细胞核Nucleus 1 1
GHNLP8D-2 GH_D02G1173 995 109.24 5.52 -0.416 细胞核Nucleus 1 1
GHNLP8D-3 GH_D10G1364 951 104.24 5.16 -0.393 细胞核Nucleus 1 1
GrNLP1-1 Gorai.002G206700.1 932 102.90 5.61 -0.433 细胞核Nucleus 1 1
GrNLP1-2 Gorai.008G041100.1 949 104.46 6.29 -0.441 细胞核Nucleus 1 1
GrNLP1-3 Gorai.005G259100.1 782 87.28 7.28 -0.381 细胞核Nucleus 1 1
GrNLP4-1 Gorai.002G115800.1 925 102.80 6.35 -0.468 细胞核Nucleus 1 1
GrNLP4-2 Gorai.009G187500.1 940 104.66 6.19 -0.488 细胞核Nucleus 1 1
GrNLP7-1 Gorai.006G206500.1 970 106.38 5.13 -0.352 细胞核Nucleus 1 1
GrNLP7-2 Gorai.004G131700.1 986 108.56 5.62 -0.338 细胞核Nucleus 1 1
GrNLP7-3 Gorai.003G074300.1 986 108.77 5.89 -0.397 细胞核Nucleus 1 1
GrNLP8-1 Gorai.005G013500.1 693 76.92 6.04 -0.457 叶绿体Chloroplast 1 1
GrNLP8-2 Gorai.005G126000.1 993 109.11 5.48 -0.431 细胞核Nucleus 1 1
GrNLP8-3 Gorai.011G138700.1 951 104.15 5.17 -0.396 细胞核Nucleus 1 1

Fig. 1

Construction of the neighbor-joining phylogenetic tree of NLP gene family members from 9 species GH: G. hirsutum; Gb: G. barbadense; Ga: G. arboreum; Gr: G. raimondii; At: A. thaliana; Os: O. sativa; Zm: Z. mays; Bna: B. napus; Bd: B. distachyon"

Fig. 2

Analysis of the phylogenetic evolution tree (A), conserved motifs (B), gene structure (C) of NLP proteins in 4 cottons and the sequences of identifed motifs (D)"

Table 2

The secondary structure of NLP proteins in cotton"

蛋白Protein α-螺旋 α-helix (%) β-转角 β-turn (%) 无规则卷曲 Random coil (%) 延长链 Extended strand (%)
GaNLP1-1 30.38 4.28 51.68 13.66
GaNLP1-2 29.51 4.56 52.34 13.59
GaNLP1-3 30.72 5.14 49.23 14.91
GaNLP4-1 26.81 4.43 55.68 13.08
GaNLP4-2 28.62 5.30 51.73 14.36
GaNLP7-1 28.12 4.02 53.04 14.83
GaNLP7-2 26.47 4.16 55.38 14.00
GaNLP7-3 25.05 3.65 57.10 14.20
GaNLP8-1 25.99 3.84 57.03 13.14
GaNLP8-2 25.48 4.45 57.43 12.64
GaNLP8-3 27.22 4.75 54.96 13.08
GbNLP1A-1 29.32 4.62 51.99 14.07
GbNLP1A-2 28.13 4.64 54.27 12.96
GbNLP1A-3 32.49 5.20 47.08 15.23
GbNLP1D-1 28.76 4.72 53.00 13.52
GbNLP1D-2 28.03 4.53 53.74 13.70
GbNLP1D-3 31.84 5.37 47.95 14.83
GbNLP4A-1 26.59 4.32 56.32 12.76
GbNLP4A-2 28.12 4.31 54.09 13.47
GbNLP4D-1 27.48 4.64 54.19 13.69
GbNLP4D-2 27.37 3.60 56.05 12.98
GbNLP7A-1 29.24 3.52 52.08 15.15
GbNLP7A-3 25.39 3.43 57.86 13.32
GbNLP7D-1 28.14 4.33 53.09 14.43
GbNLP7D-2 27.32 3.34 55.06 14.29
GbNLP7D-3 27.34 3.54 58.07 11.05
GbNLP8A-1 26.19 3.94 56.72 13.14
GbNLP8A-2 26.11 3.93 55.75 14.21
GbNLP8A-3 27.34 4.31 55.31 13.04
GbNLP8D-1 26.09 3.94 57.33 12.64
GbNLP8D-2 26.41 3.92 56.33 13.35
GbNLP8D-3 27.02 4.00 55.84 13.14
GHNLP1A-1 29.54 4.94 51.99 13.53
GHNLP1A-2 29.19 4.32 53.74 12.75
GHNLP1A-3 32.57 5.11 47.00 15.33
GHNLP1D-1 28.65 4.40 53.22 13.73
GHNLP1D-2 28.77 4.53 53.21 13.49
GHNLP1D-3 32.86 4.73 47.44 14.96
GHNLP4A-1 27.46 4.22 55.35 12.97
GHNLP4A-2 27.26 4.74 54.20 13.79
GHNLP4D-1 27.04 4.42 56.18 12.36
GHNLP4D-2 26.57 4.00 56.70 12.74
GHNLP7A-1 27.53 3.81 54.33 14.33
GHNLP7A-2 25.98 4.24 56.30 13.49
GHNLP7A-3 24.75 3.96 56.29 15.01
GHNLP7D-1 27.32 3.92 54.12 14.64
GHNLP7D-2 27.08 3.45 54.97 14.50
GHNLP7D-3 26.47 3.85 55.88 13.79
GHNLP8A-1 26.49 4.25 56.62 12.64
GHNLP8A-2 26.31 4.44 56.35 12.90
GHNLP8A-3 25.97 4.52 56.78 12.72
GHNLP8D-1 25.28 4.15 57.84 12.74
GHNLP8D-2 25.33 3.52 58.29 12.86
GHNLP8D-3 26.39 4.31 55.42 13.88
GrNLP1-1 27.58 4.51 54.51 13.41
GrNLP1-2 27.82 4.43 54.69 13.07
GrNLP1-3 32.61 5.50 47.06 14.83
GrNLP4-1 27.24 4.22 56.43 12.11
GrNLP4-2 28.83 4.04 53.40 13.72
GrNLP7-1 27.73 4.33 54.23 13.71
GrNLP7-2 25.56 3.45 56.29 14.71
GrNLP7-3 25.96 3.55 56.29 14.20
GrNLP8-1 27.27 3.61 55.70 13.42
GrNLP8-2 25.18 4.13 57.20 13.49
GrNLP8-3 26.81 4.10 55.94 13.14

Fig. 3

Chromosomal localization analysis of NLP gene family members in 4 cottons"

Table 3

The ratio of Nonsynonymous substitution (Ka) and Synonymous substitution (Ks) of NLP replication gene pairs in cotton"

基因对Gene pairs Ka/Ks 基因对Gene pairs Ka/Ks 基因对Gene pairs Ka/Ks 基因对Gene pairs Ka/Ks
GHNLP4A-1/GHNLP1A-1 0.118 GHNLP7A-3/GHNLP7D-2 0.230 GHNLP1A-2/GHNLP4D-2 0.214 GbNLP1A-1/GbNLP1D-3 0.237
GHNLP1A-1/GHNLP1A-3 0.236 GHNLP8A-1/GHNLP8D-3 0.360 GHNLP1A-2/GHNLP1D-2 0.462 GbNLP8A-1/GbNLP8D-1 0.276
GHNLP4A-1/GHNLP4A-2 0.379 GHNLP8A-2/GHNLP8D-3 0.292 GHNLP4D-1/GHNLP1D-1 0.143 GbNLP8A-2/GbNLP8D-1 0.305
GHNLP1A-1/GHNLP4A-2 0.217 GHNLP1A-3/GHNLP1A-2 0.225 GHNLP1D-1/GHNLP1D-3 0.237 GbNLP8A-2/GbNLP8D-3 0.290
GHNLP4A-1/GHNLP1A-2 0.133 GHNLP1A-3/GHNLP1D-1 0.238 GHNLP4D-1/GHNLP4D-2 0.355 GbNLP1A-3/GbNLP1D-1 0.233
GHNLP1A-1/GHNLP1A-2 0.224 GHNLP1A-3/GHNLP1D-3 0.229 GHNLP1D-1/GHNLP4D-2 0.198 GbNLP1A-3/GbNLP1D-3 0.262
GHNLP4A-1/GHNLP4D-1 0.256 GHNLP1A-3/GHNLP1D-2 0.224 GHNLP1D-1/GHNLP1D-2 0.234 GbNLP1A-3/GbNLP1D-2 0.222
GHNLP1A-1/GHNLP1D-1 0.302 GHNLP4A-2/GHNLP1A-2 0.205 GHNLP4D-1/GHNLP1D-2 0.121 GbNLP8A-3/GbNLP8D-1 0.366
GHNLP4A-1/GHNLP1D-1 0.137 GHNLP4A-2/GHNLP4D-1 0.342 GHNLP8D-1/GHNLP8D-3 0.366 GbNLP8A-3/GbNLP8D-3 0.530
GHNLP1A-1/GHNLP4D-1 0.148 GHNLP4A-2/GHNLP1D-1 0.203 GHNLP1D-3/GHNLP1D-2 0.226 GbNLP1A-2/GbNLP1D-1 0.237
GHNLP1A-1/GHNLP1D-3 0.233 GHNLP4A-2/GHNLP4D-2 0.453 GHNLP7D-3/GHNLP7D-2 0.233 GbNLP1A-2/GbNLP1D-3 0.224
GHNLP4A-1/GHNLP4D-2 0.371 GHNLP4A-2/GHNLP1D-2 0.201 GHNLP4D-2/GHNLP1D-2 0.218 GbNLP1D-1/GbNLP1D-3 0.236
GHNLP1A-1/GHNLP4D-2 0.205 GHNLP7A-2/GHNLP7D-2 0.423 GHNLP7D-2/GHNLP7D-1 0.246 GbNLP1D-1/GbNLP1D-2 0.234
GHNLP1A-1/GHNLP1D-2 0.222 GHNLP7A-1/GHNLP7D-2 0.263 GaNLP8-2/GaNLP8-3 0.285 GrNLP4-1/GrNLP1-1 0.152
GHNLP8A-1/GHNLP8A-3 0.364 GHNLP7A-1/GHNLP7D-1 0.194 GaNLP1-1/GaNLP1-3 0.234 GrNLP1-1/GrNLP1-3 0.246
GHNLP8A-2/GHNLP8A-3 0.288 GHNLP8A-3/GHNLP8D-1 0.370 GaNLP1-3/GaNLP1-2 0.219 GrNLP1-1/GrNLP1-2 0.237
GHNLP8A-1/GHNLP8D-1 0.284 GHNLP8A-3/GHNLP8D-3 0.484 GbNLP1A-1/GbNLP1A-3 0.236 GrNLP4-1/GrNLP4-2 0.348
GHNLP8A-2/GHNLP8D-2 0.277 GHNLP1A-2/GHNLP1D-1 0.235 GbNLP4A-1/GbNLP4D-1 0.274 GrNLP1-3/GrNLP1-2 0.238
GHNLP7A-3/GHNLP7D-3 0.305 GHNLP1A-2/GHNLP1D-3 0.224 GbNLP1A-1/GbNLP1D-1 0.261 GrNLP8-2/GrNLP8-3 0.286

Fig. 4

Collinearity analysis of NLP genes A: Collinearity analysis of NLP genes in G. hirsutum, G. arboreum, and G. raimondii; B: Collinearity analysis of NLP genes in G. barbadense, G. arboreum, and G. raimondii"

Fig. 5

Statistics of cis-acting elements of promoters of NLP family’s genes in cotton Each column represented a kind of cis-acting element, and each row represented a cotton NLP family gene. The numbers in the grids represented the numbers of cis-acting elements. The legend at the upper right corner represented the number range of cis-acting elements. The darker red represented the more quantity. The darker blue represented the less quantity"

Fig. 6

Expression analysis of GHNLP genes in different tissues and different stages of fiber development in G. hirsutum A: The heat map of expression levels of GHNLP genes in different tissues; B: The heat map of expression levels of GHNLP genes at ovule and fiber development"

Fig. 7

Expression analysis of GHNLP genes under different abiotic stress in G. hirsutum"

Fig. 8

PPI network of GHNLPs proteins and the expression correlation analysis of proteins interacted with NLPs A: PPI network of GHNLPs from upland cotton based on the homologous proteins in A. thaliana; B: Correlation analysis of the expression characteristics of proteins interacted with NLPs in the PPI network. The name of cotton NLP proteins are highlighted in red color"

Fig. 9

Expression analysis of GHNLPs during nitrogen starvation ***:P<0.001;**:P<0.01;*:P<0.05"

Fig. 10

Expression analysis of GHNLPs during nitrogen resupply"

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