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蚕豆VfNHX1基因克隆及初步功能验证

金文海 樊有存 李萍 范惠玲 侯万伟 滕长才 刘玉皎 武学霞

金文海,樊有存,李萍,等. 蚕豆VfNHX1基因克隆及初步功能验证 [J]. 福建农业学报,2024,39(6):689−699 doi: 10.19303/j.issn.1008-0384.2024.06.008
引用本文: 金文海,樊有存,李萍,等. 蚕豆VfNHX1基因克隆及初步功能验证 [J]. 福建农业学报,2024,39(6):689−699 doi: 10.19303/j.issn.1008-0384.2024.06.008
JIN W H, FAN Y C, LI P, et al. Cloning and Preliminary Functional Verification of VfNHX1 in Vicia faba L. [J]. Fujian Journal of Agricultural Sciences,2024,39(6):689−699 doi: 10.19303/j.issn.1008-0384.2024.06.008
Citation: JIN W H, FAN Y C, LI P, et al. Cloning and Preliminary Functional Verification of VfNHX1 in Vicia faba L. [J]. Fujian Journal of Agricultural Sciences,2024,39(6):689−699 doi: 10.19303/j.issn.1008-0384.2024.06.008

蚕豆VfNHX1基因克隆及初步功能验证

doi: 10.19303/j.issn.1008-0384.2024.06.008
基金项目: 国家自然科学基金项目(42267008);国家现代农业产业技术体系项目(CARS-08)
详细信息
    作者简介:

    金文海(1996 —),男,硕士生,主要从事作物抗逆的生理和分子机制研究,E-mail:2669455772@qq.com

    通讯作者:

    武学霞(1988 —),女,博士,副教授,主要从事作物育种及作物抗逆分子机制研究,E-mail:xuexun111@163.com

  • 中图分类号: S643.6

Cloning and Preliminary Functional Verification of VfNHX1 in Vicia faba L.

  • 摘要:   目的  探究蚕豆(Vicia faba L.)VfNHX1基因在响应盐胁迫过程中的作用。  方法  通过3′和5′RACE方法,从蚕豆中克隆了1个Na+/H+逆向转运蛋白编码基因VfNHX1,并对其进行了生物信息学分析、亚细胞定位、盐胁迫下的表达分析和初步功能验证。  结果  (1)该基因全长2255 bp,CDS编码区长1 629 bp,编码542个氨基酸;(2)生物信息学分析显示,该蛋白有10个跨膜区,不具有信号肽,是一个结构稳定的膜蛋白,且包含1个NHX 蛋白家族特有的Na-H Exchanger结构域;亚细胞定位分析显示VfNHX1定位在液泡膜上;(3)实时荧光定量PCR(qRT-PCR)分析显示,在NaCl处理后,叶片中VfNHX1表达量呈现先降低后升高,随即又下降的变化趋势,且在12 h时达最高值;根中VfNHX1表达量先降低后升高,在48 h时表达量显著升高(P<0.01);(4)酵母生长试验结果表明,VfNHX1 可以提高盐敏感酵母突变体AXT4K对高盐的耐受能力。  结论  VfNHX1基因能够响应盐胁迫,是蚕豆潜在抗盐功能基因。
  • 图  1  蚕豆中的RNA

    1:蚕豆RNA;M:DL 2000 Marker。

    Figure  1.  RNA in V. faba L.

    1: RNA in V. faba; M: DL 2000 Marker.

    图  2  蚕豆中的cDNA

    M:DL2000 Marker;1:反转录后的cDNA;2、3分别是RACE试剂盒反转录后的5′-cDNA、3′-cDNA。

    Figure  2.  cDNA in Vicia faba L.

    M: DL2000 Marker; 1: cDNA after reverse transcription; 2 and 3: 5'-cDNA and 3'-cDNA after reverse transcription of RACE kit, respectively.

    图  3  VfNHX1基因PCR扩增结果

    M:DL2000 Marker;1~2:NHX1基因中间片段; 3~4:NHX1基因5’端;5~6:NHX1基因3’端。M: DL2000 Marker; 1–2: NHX1 gene internal segment; 3–4: 5’ end of the NHX1 gene; 5–6: 3’ end of the NHX1 gene.

    Figure  3.  PCR amplification of VfNHX1

    图  4  VfNHX1蛋白进化树

    Figure  4.  Phylogenetic tree of VfNHX1 protein

    图  5  VfNHX1同源蛋白多序列比对

    以相似性 50% 为阈值,蓝色标注:相似性 ≥ 50%;粉色标注:相似性 ≥ 75%;黑色标注:相似性 ≥100%。

    Figure  5.  Multiple sequence alignment of VfNHX1 homologous protein

    With threshold at 50% similarity, blue indicates ≥50%; pink, ≥75%; black, 100%.

    图  6  VfNHX1氨基酸种类及含量

    Figure  6.  Amino acid types and contents of VfNHX1

    图  7  VfNHX1的信号肽(A)、氨基酸序列(B)、保守结构域分析(C)

    Figure  7.  Signal peptide (A), amino acid sequence (B), and conserved domain (C) of VfNHX1

    图  8  VfNHX1蛋白结构预测

    A:蛋白二级结构预测;B:蛋白三级结构测。

    Figure  8.  Predicted structure of VfNHX1 protein

    A: predicted protein secondary structure; B: predicted protein tertiary structure.

    图  9  VfNHX1的亚细胞定位

    Figure  9.  Subcellular localization of VfNHX1

    图  10  不同盐胁迫时间下VfNHX1在叶和根中的表达水平

    不同小写字母表示同一组织部位不同处理时间之间差异显著(P<0.05)。Different lowercase letters indicate significant differences among different times within the same tissue site(P < 0.05) .

    Figure  10.  Relative expressions of VfNHX1 in leaves and roots under different durations of salt stress

    图  11  盐胁迫下酵母生长

    不同小写字母代表差异显著(P<0.05)。

    Figure  11.  Yeast growth under salt stress

    Different lowercase letters represent significant difference at P<0.05.

    表  1  VfNHX1基因克隆及荧光定量PCR引物

    Table  1.   Primers for VfNHX1 cloning and qRT-PCR

    引物名称
    Primer
    引物序列
    Primer sequence (5′→3′)
    用途
    Application
    NHX1-127F CTTGAGGAGAATCGGTGGATGAA 中间片段克隆
    Intermediate fragment cloning
    NHX1-1270R GTGCTTGTGATCATGATTGCATTG 中间片段克隆
    Intermediate fragment cloning
    NHX1-5′-GSP1 CGTAAGCACTGAGCAGACCTGTCAAAACGC 5′第一轮引物
    5′ first round primers
    NHX1-5′-NGSP1 GGTGTCTCGTCTTGATTTAGCACCTGCAACG 5′第二轮引物
    5′ second round primers
    NHX1-3′-GSP2 CAAGCACTCTCCTTGGCGTTTTGACAGGTC 3′第一轮引物
    3′ first round primers
    NHX1-3′-NGSP2 CCTGTCGTTTGTTGCCGAGATCTTCATCTTCC 3′第二轮引物
    3′ second round primers
    UPM CTAATACGACTCACTATAGGGCAAGCAGTGGTATCAACGCAGAGT 3′5′第一轮引物
    3′5′ first round primers
    UPS CTAATACGACTCACTATAGGGC 3′5′第二轮引物
    3′5′ second round primers
    NHX1-438F TGATGCTACCTCAGTGGTGCTT 荧光定量PCR
    qRT-PCR
    NHX1-622R AGTGCCTGCCAATGTAGAGC 荧光定量PCR
    qRT-PCR
    ELF1A-F GTGAAGCCCGGTATGCTTGT 内参基因
    Reference gene
    ELF1A-R CTTGAGATCCTTGACTGCAACATT 内参基因
    Reference gene
    下载: 导出CSV

    表  2  生物信息学分析网站

    Table  2.   Bioinformatics analysis website

    用途 Function 网址 Website
    蛋白质信号肽分析
    Analysis of protein signal peptide
    https://services.healthtech.dtu.dk/services/signalp-6.0/
    蛋白质跨膜结构
    Protein transmembrane structure
    http://pfam-legacy.xfam.org/
    蛋白质亚细胞定位
    Protein subcellular localization
    http://www.csbio.sjtu.edu.cn/bioinf/plant-multi/
    保守结构域分析
    Conservative domain analysis
    https://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi
    蛋白质二级结构预测
    Protein secondary structure prediction
    https://npsaprabi.ibcp.fr/cgibin/npsa_automat.pl?page=npsa%20_sopma.html
    蛋白质三级结构预测
    Protein tertiary structure prediction
    https://swissmodel.expasy.org
    蛋白质理化性质
    Physicochemical properties of protein
    https://web.expasy.org/protparam/
    数据可视化
    Data visualization
    https://www.chiplot.online/
    下载: 导出CSV
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  • 收稿日期:  2024-01-03
  • 录用日期:  2024-06-02
  • 修回日期:  2024-05-28
  • 网络出版日期:  2024-07-10
  • 刊出日期:  2024-06-28

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