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作物学报

作物学报 ›› 2023, Vol. 49 ›› Issue (6): 1496-1517.doi: 10.3724/SP.J.1006.2023.24159

• 作物遗传育种·种质资源·分子遗传学 • 上一篇    下一篇

陆地棉硝酸盐转运体NRT基因家族鉴定及表达分析

马春敏1(), 李维希2, 李芳军1, 田晓莉1,*(), 李召虎1   

  1. 1中国农业大学农学院作物化控研究中心, 北京 100193
    2南京农业大学作物遗传与种质创新国家重点实验室, 江苏南京 210095
  • 收稿日期:2022-07-10 接受日期:2022-10-10 出版日期:2023-06-12 网络出版日期:2022-10-24
  • 通讯作者: *田晓莉, E-mail: tianxl@cau.edu.cn
  • 作者简介:E-mail: lmjy611@126.com
  • 基金资助:
    国家转基因生物新品种培育重大专项(2011ZX08005-004-008)

Identification and expression analysis of nitrate transporter NRT gene family in upland cotton (Gossypium hirsutum L.)

MA Chun-Min1(), LI Wei-Xi2, LI Fang-Jun1, TIAN Xiao-Li1,*(), LI Zhao-Hu1   

  1. 1Crop Chemical Control Research Center, College of Agronomy and Biotechnology, China Agricultural University, Beijing 100193, China
    2State Key Laboratory of Crop Genetics & Germplasm Enhancement, Nanjing Agricultural University, Nanjing 210095, Jiangsu, China
  • Received:2022-07-10 Accepted:2022-10-10 Published:2023-06-12 Published online:2022-10-24
  • Contact: *E-mail: tianxl@cau.edu.cn
  • Supported by:
    National Major Project for Developing New GM Crops(2011ZX08005-004-008)

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摘要：

硝酸盐转运体(nitrate transporters, NRTs)在植物氮素吸收、利用和存储等过程中发挥着重要作用。本研究利用HMM软件和Blastp方法, 在陆地棉中, 鉴定出了106个GhNRT1/PTR (NPF) (Nitrate transporter 1 (NRT1)/Peptide Transporter (PTR) family (NPF))和14个GhNRT2 (Nitrate transporter 2 family)成员, 对它们的保守结构域、系统发育关系、理化性质、亚细胞定位、保守基序、基因结构、启动子区顺式作用元件和表达模式进行了分析。结果表明, GhNPF均具有典型的PTR2 (Peptide Transporter 2 family, 肽转运蛋白)结构域, 个别蛋白(GhNPF2.6bD、GhNPF4.1cA和GhNPF2.14aD)出现了2个PTR2和/或其他结构域, 表明棉花NPF进化保守性较低; GhNRT2具有典型的MFS_1 (Major Facilitator Superfamily, 主要促进子超家族)单域。多数GhNRTs定位于细胞质膜上, 为疏水性蛋白。系统发育分析显示GhNRTs可分为10个类群, 相同类群具有相似的基因结构及基序分布。顺式作用元件组成表明, 大部分GhNRTs的表达可能与植物激素、非生物胁迫和光反应等相关。此外, GhNPF不同亚类间的表达模式存在一定差异, 但同一亚类内不同成员的表达模式相对保守; GhNRT2基因则主要在根中高表达。盐胁迫处理后的转录组数据分析发现, 近1/5的GhNRTs基因表达量发生了显著上调或下调, 表明其可能参与棉花盐胁迫应答。选择6个GhNRTs基因检测其在根系、幼叶、功能叶和老叶中的表达对不同NO3-供应水平的响应发现, GhNPF6.3dA和GhNPF7.3aA可能具有双亲和吸收NO3-的能力, GhNPF6.2bD则可能编码高亲和NO3-转运蛋白; 三者在功能叶和老叶中可能参与NO3-卸载。这些结果与拟南芥等植物中的报道不同。上述研究结果为进一步揭示棉花硝酸盐转运蛋白的功能提供了参考, 为解析棉花氮素吸收和利用机制提供了初步依据。

关键词: 陆地棉, 硝酸盐转运体, GhNPF, GhNRT2, 表达模式

Abstract:

Nitrate transporters (NRTs) play an important role in plant nitrogen absorption, utilization, and storage. In this study, 106 GhNRT1/PTR (NPF) (Nitrate transporter 1 (NRT1)/Peptide Transporter (PTR) family (NPF)) and 14 GhNRT2 (Nitrate transporter 2 family) were identified from Gossypium hirsutum L. (TM-1) by HMM software and Blastp method. The conserved domains, phylogenetic relationships, physicochemical properties, subcellular localization, conserved motifs, gene structure, promoter cis-acting elements, and expression patterns of these GhNRTs were analyzed. The results showed that GhNPF had a typical PTR2 (Peptide Transporter 2 family) domain. Two PTR2 and/or other domains were found in individual proteins (GhNPF2.6bD, GhNPF4.1cA, and GhNPF2.14aD), indicating that GhNPF was less evolutionarily conserved. The GhNRT2 had a typical MFS_1 (Major Facilitator Superfamily) domain. Most proteins were located on the cytoplasmic membrane with hydrophobic properties. Phylogenetic analysis showed that these GhNRTs could be divided into 10 groups, and the same group had similar gene structure and motif distribution. The composition of cis-acting elements indicated that the relative expression levels of most GhNRTs could be related to plant hormones, abiotic stress, and light response. In addition, the relative expression patterns of GhNPF were different among the diverse subgroups, but the relative expression patterns of different members in the same subgroup were mostly conserved. GhNRT2 genes were mainly expressed in roots. Moreover, the transcriptome data with salt stress treatment revealed that the relative levels of nearly 1/5 GhNRTs were significantly up-regulated or down-regulated, indicating that they probably function in response to salt stress. Six GhNRTs were selected to detect the response of their expression in roots, young leaves, functional leaves, and old leaves to different NO3- supply levels. The results showed that GhNPF6.3dA and GhNPF7.3aA may have the ability to absorb NO3- with dual affinity, while GhNPF6.2bD may encode high-affinity NO3- transporter. The three may be involved in NO3- unloading in functional leaves and old leaves. These results were different from those reported in plants such as Arabidopsis. In conclusion, the results provide a reference for further functional characterization of nitrate transporters and provide a preliminary basis for the mechanism analysis of nitrogen absorption and utilization in cotton.

Key words: Gossypium hirsutum L., nitrate transporters, GhNPF, GhNRT2, expression pattern

表1

RT-PCR引物序列"

基因名称
Gene name		 引物序列
Primer sequence (5'-3')
GhActin9 F: GCCTTGGACTATGAGCAGGA R: AAGAGATGGCTGGAAGAGGA
GhNPF4.6cA F: CCAATCTTCTTCTTTGGCTCTGG R: AGAAGGGCTTGCAGAGAGGTTC
GhNPF6.1aA F: GCTGCCTTCAAGAAACGACA R: GAGTGCTGCCTTATCCAAGC
GhNPF6.2bD F: CTCTTGTGTTTGCTTGGTGGC R: AATGTGCCAGTCCCAAGTGT
GhNPF6.3dA F: CCCAAATCGCTGCAGTGTTT R: CGAAACTGCTTGCTGTGAGGC
GhNPF7.3aA F: GTTGAAGAGCAGAAGAAGGGAAC R: ACCTTGCTCAACGAATAGGGATG
GhNRT2.4aA F: CCAGTGGGGTAGCATGTTTC R: GCTTTCCACGCTCAGACCG

图1

陆地棉NRT蛋白与拟南芥NRT蛋白的系统发育关系 利用MEGA 7.0中的Clustal W比对陆地棉(120条)和拟南芥(60条)的NRT蛋白序列, 采用邻接法构建系统发育树。红色星表示陆地棉NRT, 绿色三角形表示拟南芥NRT。彩色圆点代表已被验证的AtNRTs的底物, 汇总每个亚族的主要底物构成外圆环。"

图2

陆地棉(A)、拟南芥(B) NRT蛋白保守结构域分析"

表2

陆地棉NRT蛋白的理化性质及亚细胞定位预测"

基因名称
Gene ID		 分子质量
Molecular weight (kD)		 等电点
pI		 氨基酸数
Number of amino acids		 亲水性
Grand average of hydropathicity		 脂肪指数
Aliphatic index		 不稳定系数Instability index 亚细胞定位
Subcellular localization
GhNPF1.1cD 63.711 9.13 579 0.352 101.93 43.69 plas
GhNPF1.2aA 66.070 8.65 603 0.342 100.15 46.12 plas
GhNPF2.6bA 107.252 8.60 982 0.521 114.58 35.36 plas
GhNPF2.6bD 122.915 9.21 1118 0.438 110.49 37.97 plas
GhNPF2.6aA 61.214 8.61 558 0.369 107.85 34.68 plas
GhNPF2.11aD 66.821 8.91 602 0.200 96.71 30.10 plas
GhNPF2.14aD 93.651 6.76 844 0.302 103.83 39.71 plas
GhNPF2.14bD 65.256 7.88 588 0.353 104.25 35.93 plas
GhNPF3.1dA 54.147 8.45 491 0.281 106.42 37.18 plas
GhNPF3.1aD 64.861 8.48 586 0.220 100.55 30.67 plas
GhNPF3.1cD 65.818 8.37 593 0.210 100.35 32.67 plas
GhNPF4.1aA 57.776 8.52 519 0.327 96.99 34.86 plas
GhNPF4.3bD 65.985 8.46 596 0.255 97.53 35.11 plas
GhNPF4.4aD 64.965 7.52 587 0.341 98.13 37.19 plas
GhNPF4.1cA 119.341 8.99 1078 0.362 102.18 29.89 plas
GhNPF4.6bD 64.247 9.16 582 0.339 102.94 43.31 plas
GhNPF4.6cD 63.787 8.54 580 0.356 101.43 42.33 plas
GhNPF4.6aA 56.049 9.33 511 0.448 102.90 39.84 plas
GhNPF4.7aA 57.238 8.89 519 0.385 97.17 42.30 plas
GhNPF5.2aD 66.315 9.16 594 0.243 98.15 28.95 plas
GhNPF5.4bD 63.908 9.06 578 0.274 102.04 30.58 plas
GhNPF5.7aD 66.067 8.60 594 0.276 106.72 35.40 plas
GhNPF5.9aA 60.359 7.86 542 0.367 101.62 37.62 plas
GhNPF5.10aA 62.338 8.76 571 0.411 104.31 34.02 plas
GhNPF5.10dA 64.148 9.22 579 0.149 91.16 36.18 plas
GhNPF6.1aA 70.503 8.91 634 0.290 99.54 39.06 plas
GhNPF6.2bD 63.343 9.33 582 0.375 98.04 29.66 plas
GhNPF6.3bA 64.680 8.91 586 0.317 105.00 35.83 plas
GhNPF6.4bA 65.734 8.95 595 0.277 106.00 24.31 plas
GhNPF7.1bA 65.353 5.96 598 0.315 99.60 26.46 plas
GhNPF7.2aD 66.711 6.47 600 0.216 94.93 27.77 plas
GhNPF7.3aA 66.570 6.24 599 0.207 95.73 29.23 plas
GhNPF8.1aA 63.838 8.60 569 0.200 96.98 23.26 plas
GhNPF8.2cA 64.192 7.89 576 0.152 93.18 21.88 plas
GhNPF8.3bA 64.679 5.97 584 0.248 93.89 30.46 plas
GhNRT2.4bD 57.456 9.31 530 0.345 87.28 41.81 plas
GhNRT2.4aA 57.526 8.82 532 0.331 88.05 41.58 plas
GhNRT2.3aD 57.938 9.21 536 0.392 86.51 32.60 plas
GhNRT2.2aA 56.878 8.59 526 0.319 87.60 35.91 plas
GhNRT2.5aD 54.632 9.15 506 0.457 94.68 38.68 plas

图3

陆地棉GhNRTs基因在染色体上的定位"

图4

陆地棉NRT蛋白的保守基序(左)和基因结构(右)"

图5

GhNRTs启动子区域的顺式调控元件分析"

图6

GhNRTs基因在棉花不同器官和组织中的表达水平 每个基因对应的FPKM数值log2标准化后绘制基因表达热图。DPA: 开花后天数。"

图7

不同转录组中的GhNRTs基因表达分析 使用每个基因对应的FPKM数值log2标准化后绘制基因表达热图。R1: 1 cm胚根根尖; R2: 三叶期5 cm侧根; R3: 三叶期2 cm侧根; R4: 三叶期幼根; L1: 二叶期幼叶; L2、L3: 三叶期幼叶; L4: 六叶期功能叶。"

图8

GhNRTs基因对200 mmol L-1 NaCl胁迫的响应 每个基因对应的FPKM数值log2标准化后绘制基因表达热图。R: 三叶期侧根2 cm; CK: 对照。"

图9

部分GhNRTs基因表达对供氮水平的响应 采用2-ΔΔCt算法计算基因的相对表达量。将C→S处理0 h根中GhNRT2.4aA的相对表达量设为1。C→S: 7.5 mmol L-1→0 mmol L-1 NO3-; L→C: 1.39 mmol L-1→7.5 mmol L-1 NO3-; L→C→S: 1.39 mmol L-1→7.5 mmol L-1→0 mmol L-1 NO3-; R: 根; YL: 幼叶; ML: 成熟叶/功能叶(倒四叶); OL: 老叶(第1~2叶)。柱上“*”表示各部位不同时间点之间差异达0.05显著水平。"

附表1

拟南芥和水稻中NRT转运体作用底物综述"

物种
Species		 名称
Name		 先前名称
Old name		 基因名称
Gene ID		 底物
Substrates		 参考文献
Reference(s)
拟南芥
Arabidopsis thaliana
 AtNPF1.1 NRT1.12 At3g16180 NO3- (oocyte); ABA, GA, JA-Ile (yeast) [1-2]
AtNPF1.2 NRT1.11 At1g52190 NO3- (oocyte); GA, JA-Ile (yeast) [1-2]
AtNPF2.3 At3g45680 NO3- (liposome); GA (yeast) [2-3]
AtNPF2.4 At3g45700 GA, JA-Ile (yeast); Cl- (oocyte) [2,4]
AtNPF2.5 At3g45710 ABA, GA (yeast); Cl- (oocyte, yeast) [2,5]
AtNPF2.6 At3g45660 GA, JA-Ile (yeast) [2]
AtNPF2.7 NAXT1 At3g45650 NO3- (liposome); GA, JA-Ile (yeast) [2,6]
AtNPF2.9 NRT1.9 At1g18880 NO3-, 4 MTB (oocyte) [7-8]
AtNPF2.10 GTR1 At3g47960 NO3-, GA, JA-Ile, 4 MTB, 8 MTO (oocyte); GA, JA-Ile (yeast) [2,7,9-11]
AtNPF2.11 GTR2 At5g62680 4 MTB, 8 MTO, NO3-, GA (oocyte) [7,9,11]
AtNPF2.12 NRT1.6 At1g27080 NO3- (oocyte); GA (yeast) [2,12]
AtNPF2.13 NRT1.7 At1g69870 NO3-, 4 MTB (oocyte); GA, JA-Ile (yeast) [2,7,13]
AtNPF2.14 At1g69860 4 MTB (oocyte) [7]
AtNPF3.1 Nitr At1g68570 NO3-, NO2-, GA (oocyte); GA, and JA-Ile (yeast) [2,14-15]
AtNPF4.1 AIT3 At3g25260 ABA, GA, JA-Ile (yeast); GA (oocyte) [2,11,16]
AtNPF4.2 AIT4 At3g25280 ABA, GA (yeast) [2,16]
AtNPF4.5 AIT2 At1g27040 ABA (yeast) [16]
AtNPF4.6 NRT1.2/AIT1 At1g69850 NO3- (oocyte); ABA (yeast, insect cell) [2,16-18]
AtNPF5.1 At2g40460 ABA, GA, JA-Ile (yeast) [2]
AtNPF5.2 PTR3 At5g46050 Dipeptides (yeast); ABA, GA (yeast) [2,19-20]
AtNPF5.3 At5g46040 ABA (yeast) [2]
AtNPF5.5 At2g38100 NO3- (oocyte) [21]
AtNPF5.6 At2g37900 GA (yeast) [2]
AtNPF5.7 At3g53960 ABA, GA, JA-Ile (yeast) [2]
AtNPF5.10 At1g22540 NO3- (oocyte) [21]
AtNPF5.11 At1g72130 NO3- (oocyte) [22]
AtNPF5.12 At1g72140 NO3- (oocyte) [22]
AtNPF5.16 At1g22550 NO3- (oocyte) [22]
AtNPF6.2 NRT1.4 At2g26690 NO3- (oocyte) [23]
AtNPF6.3 NRT1.1 At1g12110 NO3-,auxin (oocyte) [24-26]
AtNPF7.2 NRT1.8 At4g21680 NO3- (oocyte) [27]
AtNPF7.3 NRT1.5 At1g32450 NO3-,K+ (oocyte) [28-29]
AtNPF8.1 PTR1 At3g54140 Dipeptides (oocyte, yeast); JA-Ile (yeast) [2,30-32]
AtNPF8.2 PTR5 At5g01180 Dipeptides (oocyte, yeast); ABA, GA, JA-Ile (yeast) [2,31-32]
AtNPF8.3 PTR2 At2g02040 Dipeptides (yeast); Histidine [33-34]
AtNRT2.1 At1g08090 NO3- (oocyte) [35]
AtNRT2.2 At1g08100 NO3- (oocyte) [35]
AtNRT2.4 At5g60770 NO3- (oocyte) [35]
AtNRT2.5 At1g12940 NO3- (oocyte) [35]
AtNRT2.7 At5g14570 NO3- (oocyte) [35]
水稻
Oryza sativa
 OsNPF2.2 OsPTR2 Os12g44100 NO3- (oocyte) [36]
OsNPF2.4 Os03g48180 NO3- (oocyte) [37]
OsNPF7.2 Os02g47090 NO3- (oocyte) [38]
OsNPF7.3 OsPTR6 Os04g50950 Dipeptide (yeast) [39]
OsNPF8.9 OsNRT1 Os03g13274 NO3- (oocyte) [40-41]
OsNRT2.3b Os01g50820 NO3- (oocyte) [42]
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