Scientia Agricultura Sinica ›› 2019, Vol. 52 ›› Issue (22): 3950-3963.doi: 10.3864/j.issn.0578-1752.2019.22.002

• MOLECULAR GENETICS • Previous Articles     Next Articles

Identification NADP-ME Gene of Foxtail Millet and Its Response to Stress

ZHAO JinFeng,DU YanWei,WANG GaoHong,LI YanFang,ZHAO GenYou,WANG ZhenHua,CHENG Kai,WANG YuWen(),YU AiLi()   

  1. Millet Research Institute, Shanxi Academy of Agricultural Sciences/Shanxi Key Laboratory of Genetic Resources and Breeding in Minor Crops, Changzhi 046011, Shanxi
  • Received:2019-07-03 Accepted:2019-09-22 Online:2019-11-16 Published:2019-11-16
  • Contact: YuWen WANG,AiLi YU E-mail:gzswyw@163.com;yuailimail@126.com

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Abstract:

【Objective】 The objectives of this study are to identify the NADP-ME family genes (SiNADP-MEs) from foxtail millet (Setaria italica) genome, study the response of different members to abiotic stress, and to lay a theoretical foundation for revealing the role of SiNADP-ME genes in stress signal pathway of foxtail millet. 【Method】 The members of NADP-ME family in the foxtail millet genome were identified by bioinformatics methods. The protein and gene sequence of identified members were analyzed using software such as GSDS2.0, plantCARE, Clustalx, MEGA6.0 and the website ExPASy. Real-time quantitative PCR (qRT-PCR) was used to detect the expression levels of SiNADP-ME genes under different stresses at seedling stage, under drought and different light intensities stress at different growth stages. 【Result】 The NADP-ME family of foxtail millet consists of seven members, which were unevenly distributed on chromosomes 2, 3, 5, and 7 of foxtail millet. Conservative functional domain analysis revealed that all seven genes contain the conserved characteristic domains of NADP-ME. Amino acid sequence alignment revealed that the sequences were very conserved and the similarity was very high among the members. The sequence identity of 7 SiNADP-ME members was 77.30%, while the identity of NADP-MEs in different species was 56.52%. Sequence analysis showed that the sequences of SiNADP-ME1, 4, 5, and 6 were longer, encoding 576, 639, 652, and 636 amino acids residues respectively, while the sequences of SiNADP-ME2, 3, and 7 were shorter, encoding 213, 265 amino acids residues respectively. Gene structure analysis showed that SiNADP-ME1 has two alternative transcript, SiNADP-ME5 has three alternative transcript, and other genes have no alternative transcript. SiNADP-ME1, 2, 3 and 7 contain fewer introns, while SiNADP-ME4, 5 and 6 contain more introns. The prediction of protein parameters showed that the molecular weight span among members is large, ranging from 161.94 to 725.43 kD, the isoelectric point from 5.32 to 8.05, the instability index from 23.01 to 45.01, the aliphatic index from 89.19 to 107.77, and the grand average of hydropathicity from -0.218 to 0.004. Subcellular localization predictions show that SiNADP-ME members are mainly localized in chloroplasts, mitochondrial and cytoplasmic. Cis-elements analysis revealed that hormonal, stress, light, and other growth-related cis-elements are present in the promoter region of the SiNADP-ME members. Cluster analysis revealed that SiNADP-ME genes were present before the isolation of monocotyledonous and dicotyledonous plants. The homologous pairs of different species present in the phylogenetic tree revealed that they may evolve from a common ancestor, suggesting that they may have similar functions in certain signaling pathways. Stress expression analysis of seedling stage showed that the expression levels of all the SiNADP-ME family genes were significantly induced under the four stresses applied in this paper. The highest relative expression levels of SiNADP-ME1 under ABA, low temperature and NaCl treatment were 460.53, 411.50 and 15.24 folds than that of the control respectively, while the highest relative expression levels of SiNADP-ME6 under ABA, low temperature, PEG and NaCl treatment were 211.13, 15.21, 772.41 and 643.99 folds than that of the control respectively. Further analysis showed that the expression levels of SiNADP-ME1 and SiNADP-ME6 were up-regulated under drought stress at jointing, heading and filling stage.【Conclusion】 Seven members of NADP-ME gene family were identified from foxtail millet genome. All SiNADP-ME genes contain the typical characteristic domains of the NADP-ME, and their sequences are very conserved. All of the SiNADP-ME genes are involved in plant abiotic stress response, especially SiNADP-ME1 and SiNADP-ME6 may play an important role in response to abiotic stress.

Key words: foxtail millet, NADP-ME, gene expression, abiotic stress

Table 1

Primers used in this study"

基因 Gene 正向引物 Forward primer (5′-3′) 反向引物 Reverse primer (5′-3′)
SiNADP-ME1 AGGAGCATCCAGGTCATCGT ACTCGTCGTTGAGCAAGGTCT
SiNADP-ME2 AAAATTTATGGTAGATGTCAGGTCC TGACTACCTTCTGCCAAATCCT
SiNADP-ME3 TTACACACCGACTGTTGACGAG TGAGCACCCAAATCTCCAAGT
SiNADP-ME4 CTCCGATTGAAGAGTGCCG TGTGAATGCCCTTCCAACTC
SiNADP-ME5 GCAAGATATGGCACAACTCACC CCAGCACCCAGGAATAGGAA
SiNADP-ME6 ACCTATCATCCTCGCTCTATCAA CAAATGGGCTTCCACTTCC
SiNADP-ME7 AAATTTTATGGTAGATGTCAGGTCC TTTCATCCACTGGTGAGAACTTC
β-Actin GGGCAGTTGCTATCCACATC ATCACGCCACAAGACCAGAC

Table 2

Parameters of predicted foxtail millet NADP-ME genes"

基因
Gene		 基因 ID
Gene ID		 染色体位置
Chr. location		 基因长度
Genome
length
(bp)		 氨基酸
数目
Number of
amino acid		 等电点
pI		 分子量
Molecular weight
(kD)		 内含子数Intro number 不稳定
指数Instability index		 脂肪系数Aliphatic index 平均疏水指数
Grand average of hydropathicity
SiNADP-ME1 Seita.3G109300 278989-7282388 3400 576 5.60 637.72 8 39.31 91.46 -0.176
SiNADP-ME2 Seita.3G284800 26806748-26808875 2128 213 5.47 233.24 6 32.64 91.60 -0.209
SiNADP-ME3 Seita.4G119800 12185662-12187990 2329 265 5.32 296.11 8 38.32 107.77 0.004
NSiADP-ME4 Seita.5G134300 11688238-11693473 5236 639 6.31 700.38 19 45.01 89.19 -0.138
SiNADP-ME5 Seita.5G301800 35587449-35592805 5357 652 6.28 725.43 19 44.52 89.49 -0.218
SiNADP-ME6 Seita.5G314300 36545367-36550031 4665 636 8.05 698.51 18 42.98 92.25 -0.133
SiNADP-ME7 Seita.7G040900 12423964-12426409 2446 149 5.40 161.94 4 23.01 97.52 -0.054

Fig. 1

Gene structure of SiNADP-ME genes"

Table 3

Prediction of subcellular localization of SiNADP-ME protein"

基因 Gene 预测位置 Localization 可信度 Reliability
SiNADP-ME1 细胞质Cytoplasmic ;叶绿体Chloroplast ;线粒体Mitochondrial 2.254;1.083;0.957
SiNADP-ME2 细胞质Cytoplasmic ;细胞外Extracellular ; 细胞核Nuclear 3.030;0.452;0.401
SiNADP-ME3 细胞质Cytoplasmic ;线粒体Mitochondrial ;叶绿体Chloroplast 1.834;0.753;0.752
SiNADP-ME4 叶绿体Chloroplast ;线粒体Mitochondrial ;细胞质Cytoplasmic 2.491;1.274;0.390
SiNADP-ME5 线粒体Mitochondrial ;叶绿体Chloroplast ;过氧化物酶体Peroxisomal 1.463;1.380;0.829
SiNADP-ME6 线粒体Mitochondrial ;叶绿体Chloroplast ;过氧化物酶体Peroxisomal 2.206;1.616;0.449
SiNADP-ME7 细胞质Cytoplasmic;细胞外Extracellular;细胞核Nuclear 1.351;1.144;0.598

Table 4

Putative cis-elements in the promoter of SiNADP-MEs"

顺式元件
Cis-element		 典型序列
Typical sequence		 特性
Characteristic		 基因
Gene
ABRE ACGTG 脱落酸响应 Abscisic acid responsiveness SiNADP-ME1,2,4,5,6,7
CACGTG SiNADP-ME2,4,6
GCCGCGTGGC SiNADP-ME1,4,6
TCA-element TCAGAAGAGG 水杨酸响应 Salicylic acid responsiveness SiNADP-ME4,7
CGTCA-motif CGTCA 茉莉酸甲酯响应 MeJA-responsiveness SiNADP-ME1,3,4,5,6,7,
TGACG-motif TGACG SiNADP-ME3,4,5,6,7
P-box CCTTTTG 赤霉素响应 Gibberellin-responsive element
 SiNADP-ME1,3,6
GARE-motif TCTGTTG SiNADP-ME3,
TATC-box TATCCCA SiNADP-ME6
TGA-element AACGAC 生长素响应 Auxin-responsive element SiNADP-ME1,5
LTR CCGAAA 低温响应 Low-temperature responsiveness SiNADP-ME4,5
MBS CAACTG 干旱响应 Drought-inducibility SiNADP-ME3,4,5,,7
TC-rich repeats GTTTTCTTAC 防御和应激反应响应 Defense and stress responsiveness SiNADP-ME6
G-box CACGTT 光响应 Light responsiveness SiNADP-ME4,5,7
CACGTG SiNADP-ME2,4,6
CACGTC SiNADP-ME1,2,4
TACGTG SiNADP-ME1,2,6
Sp1 GGGCGG 光响应 Light responsive element SiNADP-ME2,6,7
GT1-motif GGTTAA SiNADP-ME1,2,4,5
3-AF1 binding site TAAGAGAGGAA SiNADP-ME2,3
TCCC-motif TCTCCCT 光响应 Part of a light responsive element SiNADP-ME2,4,7
GATA-motif AAGGATAAGG SiNADP-ME2,3
GATAGGG SiNADP-ME5,6
GC-motif CCCCCG 缺氧特异性诱导 Anoxic specific inducibility SiNADP-ME1,2,4,7
CAT-box GCCACT 分生组织响应 Meristem expression SiNADP-ME1,2,4,6,7
ARE AAACCA 厌氧响应 Essential for the anaerobic induction SiNADP-ME4,5
O2-site GATGACATGG 玉米蛋白代谢调控 Zein metabolism regulation SiNADP-ME1,4,5
CCAAT-box CAACGG MYBHv1结合位点 MYBHv1 binding site SiNADP-ME1,5
RY-element CATGCATG 种子特异性调控 Seed-specific regulation SiNADP-ME1,4
MSA-like (T/C)C(T/C)AACGG(T/C)(T/C)A 细胞周期调控 Cell cycle regulation SiNADP-ME1
circadian CAAAGATATC 昼夜节律 Circadian control SiNADP-ME3
motif I gGTACGTGGCG 根特异性 Root specific SiNADP-ME6

Fig. 2

Amino acid sequence alignment of NADP-MEs OsNADP-ME2：水稻Oryza sativa,XP_015640686;TaNADP-ME1：小麦Triticum aestivum,ABW77317;ZmNADP-ME2：玉米Zea mays,XP_008656303;SbNADP-ME1：高粱Sorghum bicolor,XP_002440734;ZmNADP-ME1：玉米Zea mays,NP_001150965;OsNADP-ME1：水稻Oryza sativa,BAA03949;TaNADP-ME2：小麦Triticum aestivum,CDM81737;AtNADP-M2：拟南芥Arabidopsis thaliana,NP_178093;AtNADP-M1：拟南芥Arabidopsis thaliana,NP_197960;SbNADP-ME2：高粱Sorghum bicolor,XP_021312641;PpNADP-ME1：小立碗藓Physcomitrella patens,PNR56966;PpNADP-ME2：小立碗藓Physcomitrella patens,XP_024371400;PtNADP-ME1：北美云杉Picea sitchensis,AEX13395;PtNADP-ME2：北美云杉Picea sitchensis,AEX13397。The amino acids with an entire homology are shown by a black background, and those shared non-identical conserved identity by a gray background (≥ 60% similarity)"

Fig. 3

Phylogenetic relationships of NADP-ME proteins from different species"

Fig. 4

Expression profile of SiNADP-ME gene in foxtail millet Light green,dark green, black, light red and dark red are used to represent gene expression levels. Green indicates weak gene expression, red indicates strong gene expression"

Fig. 5

Relative expression of SiNADP-ME1 and SiNADP-ME6 under normal and drought conditions during different growth stages Single asterisk show the discrepancy on 0.05 levels notable and double asterisk show the discrepancy on 0.01 levels notable. The same as below"

Fig. 6

Relative expression of SiNADP-ME1 and SiNADP-ME6 under light and drought conditions"

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