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作物学报

作物学报 ›› 2024, Vol. 50 ›› Issue (3): 576-589.doi: 10.3724/SP.J.1006.2024.31025

所属专题： 小麦：遗传育种·种质资源·分子遗传学

• 作物遗传育种·种质资源·分子遗传学 • 上一篇    下一篇

小麦TaSPX1基因的克隆、表达及耐低氮逆境的功能研究

张宝华1,2(), 刘佳静1,2, 田晓1,2, 田旭钊1,2, 董阔1, 武郁洁1, 肖凯3,*(), 李小娟1,2,*()   

  1. 1河北农业大学生命科学学院, 河北保定 071001
    2河北省植物生理和分子病理学重点实验室, 河北保定 071001
    3河北农业大学农学院, 河北保定 071001
  • 收稿日期:2023-04-06 接受日期:2023-09-13 出版日期:2024-03-12 网络出版日期:2023-10-09
  • 通讯作者: *肖凯, E-mail: xiaokai@hebau.edu.cn; 李小娟, E-mail: lxjlixiaojuan@126.com
  • 作者简介:E-mail: zbh1383306@163.com
  • 基金资助:
    国家自然科学基金项目(32071935)

Cloning, expression, and functional analysis of wheat (Triticum aestivum L.) TaSPX1 gene in low nitrogen stress tolerance

ZHANG Bao-Hua1,2(), LIU Jia-Jing1,2, TIAN Xiao1,2, TIAN Xu-Zhao1,2, DONG Kuo1, WU Yu-Jie1, XIAO Kai3,*(), LI Xiao-Juan1,2,*()   

  1. 1College of Life Sciences, Hebei Agricultural University, Baoding 071001, Hebei, China
    2Key Laboratory of Plant Physiology and Molecular Pathology, Baoding 071001, Hebei, China
    3College of Agronomy, Hebei Agricultural University, Baoding 071001, Hebei, China
  • Received:2023-04-06 Accepted:2023-09-13 Published:2024-03-12 Published online:2023-10-09
  • Contact: *E-mail: xiaokai@hebau.edu.cn; E-mail: lxjlixiaojuan@126.com
  • Supported by:
    National Natural Science Foundation of China(32071935)

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摘要：

包含SPX、SPX-EXS、SPX-MFS和SPX-RING四个亚族的植物SPX基因家族在磷信号应答中发挥重要功能, 但迄今对小麦的该家族基因成员功能了解尚少。本研究前期从小麦(Triticum aestivum)中鉴定得到一个SPX亚族成员基因TaSPX1 (GenBank No. Ak332300), 亚细胞定位分析发现其定位于细胞核。对TaSPX1和来自小麦、拟南芥和水稻SPX家族的同源蛋白进行系统进化分析, 结果表明, 其与水稻SPX亚族的OsSPX1亲缘关系较近。应用RT-qPCR技术研究发现, TaSPX1的表达量在低氮胁迫下显著增加。构建烟草(Nicotiana tabacum)过表达转基因系(overexpression lines, OE), 应用MS营养液培养对野生型(WT)和OE株系OE3和OE4植株表型进行鉴定。发现在低氮胁迫下, OE3和OE4较WT表现明显的生长优势, 植株鲜重、根重和叶面积显著增加; 包括光合速率、胞间CO2浓度、气孔导度和蒸腾速率在内的光合参数, 以及氮含量、可溶性糖、可溶性蛋白和叶绿素含量也都较WT显著增加。对氮吸收和同化相关基因的表达和酶活性测定结果表明, 上述基因的部分成员在OE植株中的表达量和氮同化酶活性升高。此外, 对包括SOD、POD和CAT在内的植株活性氧清除相关酶活力和MDA含量测定表明, 与WT相比, OE植株中保护酶的活性均明显提高, MDA含量降低。同时发现OE植株中部分保护酶基因成员的表达水平也较WT明显升高。这些结果初步证实了TaSPX1通过改善光合参数、增强氮吸收和转运以及加强保护酶系统等在介导植株抵御低氮胁迫中发挥重要作用。本项研究对小麦SPX家族成员抵御非生物逆境功能增加了新认识, 为作物抗低氮营养逆境的遗传改良提供了理论依据。

关键词: 小麦, TaSPX1, 亚细胞定位, 低氮胁迫, 功能分析

Abstract:

The SPX gene family includes four subgroups: SPX, SPX-EXS, SPX-MFS, and SPX-RING, which play an important role in phosphate signal response, but so far, little is known about the functions of this family in wheat. Previously, we identified a gene TaSPX1 (GenBank No. Ak332300), belonged to SPX subfamily from wheat (Triticum aestivum). Subcellular localization analysis showed that it targeted onto nucleus. Phylogenetic tree of TaSPX1 and its homologous proteins from the wheat, Arabidopsis, and rice SPX families showed that it was closely related to OsSPX1, a member of rice SPX subfamily. The relative expression level of TaSPX1 significantly increased under low nitrogen (low-N) stress when investigated by RT-qPCR. Transgenic tobacco (Nicotiana tabacum) overexpression lines were generated. Using the culture methods of Murashige & Skoog (MS) hydroponic solution, the phenotype of WT and OE under low-N stress treatment was investigated. We found that the plants of OE3 and OE4, two OE lines overexpressing TaSPX1, displayed increased growth vigor and leaf area, together with the enhanced plant fresh weight and root weight, and elevated photosynthetic parameters including photosynthetic rate (Pn), intercellular carbon dioxide concentration (Ci), stomatal conductance (Gs), and transpiration rate (Tr), along with the increased contents of nitrogen, soluble sugar, soluble protein, and chlorophyll content upon low-N stress with respect to WT. Studies on transport and assimilation related parameters showed that under low-N stress, the relative expression level of some related genes and the activities of nitrogen assimilation-related enzymes were increased. Assays on the SOD, POD, and CAT, the enzymes functional as cellular protector, revealed the higher activities of them in OE plants than those in WT. On the contrary, MDA content was decreased. Further RT-qPCR analysis indicated the expression levels of several protection enzymes mentioned above were higher in OE plants than those of WT under low-N stress. Therefore, TaSPX1 played an important role in mediating plant resistance to low-N stress by improving photosynthetic parameters, enhancing nitrogen absorption and transport, and strengthening the protective enzyme system. The results enrich new understanding on the function of wheat SPX family members involved in abiotic stress, and provide a theoretical basis for genetic improvement of crops against low-N stress.

Key words: wheat, TaSPX1, subcellular localization, low nitrogen stress, functional analysis

表1

用于RT-qPCR的引物"

引物名称
Primer name		 登录号
Accession number		 上游引物
Forward sequence (5′-3′)		 下游引物
Reverse sequence (5′-3′)
Ntaction U91563 ATTACGAGGATGAAGAGGAAGC TAAAAGCCTGAAGTTTGAAGA
Tatubulin U76558 CATGCTATCCCTCGTCTCGACCT CGCACTTCATGATGGAGTTGTAT
TaSPX1 Ak332300 ACTGGGGCTCTTATCCGTCT CAGCCTGCTCGATCACAGAT
NtSOD1 KJ874395 GGACAAGGAGTTCCGGATGATT CCATATTGACTTGAGCCAAGCG
NtSOD2 EU123521 TTGGGGAAGATGGTACTGCATC TCATCAGGATCAGCGTGAACAA
NtFeSOD KF724056 CAGGCCTGGAATCATCAGTT TTTCACCAAGGCAAGCTTTT
NtMnSOD1 X14482 ACCACCAGAATCACCATCAGAC CTATGCAATTTGGCGACGGTAG
NtMnSOD2 AB093097 AAGCCCTTGAACAGCTACATGA TCGAGTGGTTAATGTGACCTCC
NtCAT NTU07627 CAAGGATCTCTACGACTCGATT CTTGAGGGCAAATAATCCACCT
NtCAT1 EF532799 TCGGAGGATAAGCTTCTCCAGA ATGAGCACACTTGGGAGCATTA
NtCAT1;1 NTU93244 CAATGCTTGCCGATTTCTCT AGCAGGATCGGTATGGTCAG
NtCAT1;2 HF564632 GAAGTTCCCTGACATGGTCCAT AAGTGAACATGTGCAGGCTTTC
NtCAT1;3 HF564631 TGGCAAACGAGAGAAGTGTG AAGCAAGCTTTTGACCCAGA
NtCAT3 HF564633 GACCCCAGAGGATTTGCTGTAA ACCATGTCAGGGAACTTCATCC
NtPOD1;1 L02124 ATTAGGCTAAAGGTCCGTCGTG GCAAGTGTTTCAAAGGGGCTAG
NtPOD1;2 AB044154 GAAGGTTCAGACGCTGAGAGAA CAGGAAACAACTCCAGGACAGA
NtPOD1;3 AB044153 ATGGTCGTGGAGTTCTGGAATC GAATGTCAACCCAAGCAATCCC
NtPOD1;4 D11396 TCCTAATGTAGGTGCAGGAGGA CTCCAATTTCAGATGCAAGGGC
NtPOD1;5 AB178953 AGACCTCACCACCCAACAAC GCATCTCTTCTCCCAAGTGC
NtPOD1;6 AB027753 CTTACTGGTGCGCACACTATTG GCTGAGGAAGAAAGGTTGCATC
NtPOD1;7 AB027752 AATGGGTGCTTCTCTTCTTCGT CCCTTTTCTCTCCTGTGAAGCT
NtPOD4 AY032675 TTCCATCAGGGGATTTGAAG TGGTGCAGGAATGTTTGTGT
NtPOD9 AY032674 GAAACCCTAGCCAACCTTCC TCGTGTTCGTGGAGTTTGAG
NtNRT1.1-s AB102805 TACCGGTTTGTCGACGTGTC TCTCTTCTCCTTGTACACATAC
NtNRT1.1-t AB102806 CCGGCTTCATTGACACTCTT CCTCTTCTCCTTGTACACATAC
NtNRT1.2-s AB102807 GGGTTATCGTTCCCATTTGTCG TCAGCAAGTCTCTTCTCCTTGT
NtNRT1.2-t AB102808 GCCCTAACAGAGGTTAAGAGG TCCCCATTTCAGCAAGTCTC
NtNRT2.1 AJ557583 TAGCCGTCACATTCATGATCCT GATCGGCAGTTCTCGGCGAA
NtNRT2.2 AJ557584 CGTCGATCGTTAGGTATAATC ATTAACTACTCACACTTGGGTAA
NtNR1 X06134 ATCAGGTGGATGGATGGCGA ACAACCAACTCGAAGTACCC
NtNR2 X14059 ATGACTGGACCGTGGAAGTC AAAACACCGCACCGTTTTAG
NtNR3 JN384020 GGACGAAGGTACCGCTGATA GACTTCCACGGTCCAGTCAT
NtNR4 JN384019 TGTTTGAGCATCCGACTCAAC GGAGGTGGTCCACAAGCCA
NtNR5 XM_016606384 ATGGATATTACCGGCCGGCA TTACGTACCATCAGCATTAACA
NtNR6 XM_016583028 TTGAGGCACTGCTCAAAGAGC TCCCAAATGTGAATCACTCCC
NtNIR1 NM_001324935 ATGGCATCTTTTTCTGTTAAAT TTAATCTTCTGCTTCTTCTCTT
NtNIR2 X66145 GGGTTCCAGCTGATGATGTT TGGCATTCTCTTCTCGACCT
NtNIR3 X66147 TGGCAATTCACGAGGCAATC TCGTGGAACTGCACCAAAGT
NtNIR4 XR_001649867 AGTTGGGTTGACCAGTTTGC ACACGGATTCCACTTCCTTG
NtNIR5 XM_016583028 AGCATCTCCAGAAGACTTGG CCCAAATGTGAATCACTCCC
NtGS1 NM_001325250 ATTATGTCTCCGCTTTCAGATC ATTGAGCAAACCAGAAACAAGC
NtGS2 X95932 TCTTGGTGGTTTTCCTGGAC TGCTGAAATGCCAACTGAAG
NtGS3 XM_016584731 AAGATGGCTCAGATCTTGGCTC CTTTAAACATTCAATGCGAGCT
NtGS4 XM_016579636 GATGTGCAAACCCACACCTT CTGCAAATTCCTCGTCTGGT
NtGS5 XM_016640903 CGGGCATGATCAGAATTATTC AGATTGGTCGGGATATGAACC
NtGS6 XM_016640901 AACTAGGCTGGGATTGACG AGTCCCTCTCAGGCTCACAA
NtGS7 XM_016631331 CATTATGTCTCTGCTTTCAGAT TCATGGCAGTAACGACGTATGG

图1

TaSPX1与小麦、水稻和拟南芥SPX家族同源蛋白的系统进化分析 SPX蛋白序列分别来自小麦(TaSPXs)、水稻(OsSPXs)和拟南芥(AtSPXs), 灰色圆点为TaSPX1。"

图2

转化烟草表皮细胞的TaSPX1-GFP亚细胞定位"

图3

小麦TaSPX1基因在低氮胁迫下的表达特征 ** 表示在0.01概率水平差异显著。"

表2

TaSPX1基因启动子的顺式作用元件分析"

顺式元件
Cis-element		 目标序列
Target sequence (5′-3′)		 数目
Number		 功能
Function
TATA-BOX TATATA 23 胁迫响应元件和核心启动元件
Stress response elements and core promoter element
CAAT-BOX CAAT 39 MYBHv1结合位点
MYBHv1 binding site
MYC CATGTG/CATTTG 4 干旱和盐响应元件
Drought and salt response element
MYB CAACAG/CAACTG/CAACCA 2 干旱和盐响应元件
Drought and salt response element
ABRE TACGTGTC/ACGTG 7 ABA响应元件
ABA-responsive element
Root motif ATATT 2 根系生长
Root growth
G-box CACGTT/TACGTG/GCCACGTGGA 4 参与光响应顺式作用调节元件
cis-acting regulatory element involved in light responsiveness
LTRE AGTCGG 2 低氮胁迫响应元件
Response to low nitrogen stress
/ NANGAG 4 低氮胁迫响应元件
Response to low nitrogen stress

图4

过表达烟草植株中TaSPX1的表达分析 **表示与WT相比在0.01概率水平差异显著。"

图5

WT、OE3和OE4在低磷和低氮下的表型特征及低氮处理下的生理生化特征 A: 生长情况; B: 全株表型特征(标尺为9 cm); C: 鲜重; D: 根重; E: 叶面积; F: 叶绿素含量; G: 蒸腾速率; H: 光合速率; I: 气孔导度; J: 胞间CO2浓度。*表示与WT相比在0.05概率水平差异显著, **表示与WT相比在0.01概率水平差异显著。"

图6

低氮处理下WT、OE3和OE4植株氮含量(A)、可溶性糖(B)和可溶性蛋白(C) ** 表示与WT相比在0.01概率水平差异显著。"

图7

低氮胁迫下WT、OE3和OE4株系的氮吸收同化酶活性和基因表达 A: NIR酶活性; B: NR酶活性; C: GS酶活性; D: NRTs的相对表达量; E: NIRs的相对表达量; F: NRs的相对表达量; G: GSs的相对表达量。**表示与WT相比在0.01概率水平差异显著。"

图8

低氮胁迫下WT、OE3和OE4株系的保护酶活性、MDA含量和保护酶基因相对表达量 A: SOD酶活性; B: POD酶活性; C: CAT酶活性; D: MDA含量; E: SOD基因相对表达量; F: POD基因相对表达量; G: CAT基因相对表达量。**表示与WT相比在0.01概率水平差异显著。"

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