蚕豆<i>VfNHX1</i>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

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

金文海^{1, 2,},
樊有存^{1, 2},
李萍³,
范惠玲⁴,
侯万伟³,
滕长才³,
刘玉皎^{1, 3},
武学霞^{1, 5, ,}

1.
青海大学省部共建三江源生态与高原农牧业国家重点实验室，青海　西宁　810016
2.
青海大学生态环境工程学院，青海　西宁　810016
3.
青海大学农林科学院，青海　西宁　810016
4.
河西学院农业与生物技术学院，甘肃　张掖　734099
5.
青海师范大学高原科学与可持续发展研究院，青海　西宁　810016

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
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出版历程
- 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

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

JIN Wenhai^{1, 2
,},
FAN Youcun^{1, 2},
LI Ping³,
FAN Huiling⁴,
HOU Wanwei³,
TENG Changcai³,
LIU Yujiao^{1, 3},
WU Xuexia^{1, 5
, ,}

1.
Key Laboratory of Plateau Ecology of the Province and Qinghai Agriculture University, Xining, Qinghai　810016, China
2.
College of Eco-Environmental Engineering, Qinghai University, Xining, Qinghai　810016, China
3.
Academy of Agricultural and Forestry, Qinghai University, Xining, Qinghai　810016, China
4.
College of Agriculture and Biotechnology, Hexi University, Zhangye, Gansu　734099, China
5.
Academy of Plateau Science and Sustainability, Qinghai Normal University, Xining, Qinghai　810016, China

摘要

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。
- 蚕豆 /
- Na⁺/H⁺逆向转运蛋白 /
- 基因克隆 /
- 盐胁迫 /
- 功能验证
Abstract: Objective Role of VfNHX1 of faba bean in response to salt stress was studied. Methods A Na⁺/H⁺ reverse transporter protein-encoding gene, VfNHX1, was cloned from Vicia faba L. by 3' and 5' RACE for a bioinformatics analysis, subcellular localization determination, expression under salt stress, and a preliminary verification on the function. Results (1) VfNHX1 sequence was 2255 bp with a CDS coding region of 1629 bp encoding 542 amino acids. (2) As a stable transmembrane protein, VfNHX1 had 10 transmembrane regions without signal peptide and contained a typical Na-H exchanger conserved functional domain of NHX family. It was in the vacuolar membrane. (3) In the leaves, the expression of VfNHX1 under NaCl stress determined by qRT-PCR showed a trend of decreasing, then increasing, followed by decreasing with a peak reached in 12 h. In the roots, the expression declined initially and then rose significantly at 48 h (P＜0.01). (4) The presence of VfNHX1 in a yeast growth experiment indeed heightened the NaCl tolerance of the salt-sensitive yeast mutant AXT4K. Conclusion VfNHX1 in V. faba L. was able to respond to salt stress and could be a functional gene to boost the salt resistance of the plant.
- Vicia faba L. /
- Na⁺/H⁺ antiporter /
- cloning /
- salt stress /
- functional verification

HTML全文

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.

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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.

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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

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图 4 VfNHX1蛋白进化树

Figure 4. Phylogenetic tree of VfNHX1 protein

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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%.

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图 6 VfNHX1氨基酸种类及含量

Figure 6. Amino acid types and contents of VfNHX1

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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

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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.

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图 9 VfNHX1的亚细胞定位

Figure 9. Subcellular localization of VfNHX1

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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

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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.

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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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