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

蚕豆VfNHX1基因克隆及初步功能验证

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（42267008），国家现代农业产业技术体系（CARS-08）

详细信息

作者简介:
金文海（1996 —），男，在读硕士生，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

Cloning and preliminary functional verification of VfNHX1 in faba bean (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.
State Key Laboratory of Plateau Ecology and Agriculture Qinghai 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: 目的为探究蚕豆（Vicia faba L.）VfNHX1基因在响应盐胁迫过程中的作用。方法本研究通过3′和5′RACE方法，从蚕豆中克隆了一个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。结果显示：（1）该基因全长2255 bp，CDS编码区长1629 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，且包含一个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时达到最高值；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）酵母生长实验表明，VfNHX1 可以提高盐敏感酵母突变体AXT4K对高盐的耐受能力。结论这些结果表明，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 ] To investigate the role of the faba bean (Vicia faba L.) VfNHX1 gene in response to salt stress. Methods In this study, a Na⁺/H⁺ reverse transporter protein-encoding gene, VfNHX1, was cloned from faba bean by 3' and 5' RACE, and was subjected to bioinformatics analysis, subcellular localization, expression analysis under salt stress, and preliminary functional validation. Results (1) The results showed that the gene VfNHX1 has the sequence size of 2255 bp and the CDS coding region was 1629 bp which encodes a protein with 542 amino acids. (2) The bioinformatics analysis indicated that VfNHX1 had 10 transmembrane regions and no signal peptide. It was a stable transmembrane protein and contained a typical Na-H Exchanger conserved functional domain of NHX family. Subcellular localization analysis showed that VfNHX1 was present in vacuolar membrane. (3) The expression level of VfNHX1 after NaCl treatment was determined using real-time fluorescence quantification (qRT-PCR). The result showed that the expression of VfNHX1 in leaves showed a trend of decreasing and then increasing, followed by decreasing, and reached the highest value at 12 h. In roots, the expression of VfNHX1 decreased and then increased, and the expression was significantly increased at 48 h (P＜0.01). (4) Yeast growth experiments showed that VfNHX1 could improve the tolerance of salt-sensitive yeast mutant AXT4K to high salt stress. Conclusion These results suggested that the VfNHX1 gene is able to respond to salt stress and is a potential functional gene for salt resistance in faba bean.
- Vicia faba L. /
- Na⁺/H⁺antiporter /
- gene cloning /
- salt stress /
- functional verification

HTML全文

图 1 蚕豆中的RNA

Figure 1. RNA in Vicia faba L.

M: Marker, DL2000；1: RNA.

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图 2 蚕豆中的cDNA

M: Marker, DL2000；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: Marker, DL2000；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基因的中间片段、5′端和3′末端

M: Marker, DL2000；NHX1中间: Intermediate fragment of VfNHX1 gene；

Figure 3. The middle fragment, 5 ' end and 3 ' end of VfNHX1

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图 4 VfNHX1蛋白进化树

自展值：Bootstrap value;

Figure 4. Phylogenetic tree of VfNHX1 protein

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图 5 VfNHX1同源蛋白多序列比对

注：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%；蚕豆：Vicia faba；银豆：XP_028770753.1 (Prosopis alba)；灰木豆：XP_054802326.1 (Prosopis cineraria)；紫花苜蓿：AAR19085.1 (Medicago sativa)；镰刀紫花苜蓿：ADB27460.1 (Medicago sativa subsp) ；花生: XP_016175144.1 (Arachis ipaensis)；花生：ADK74832.1 (Arachis hypogaea)；杜兰花：XP_015939959.1 (Arachis duranensis)；木豆：XP_020209643.1 (Cajanus cajan)；大豆：NP_001237166.2 (Glycine max)；珠美海棠： ADB80440.1 (Malus zumi)；甜樱桃：XP_021817479.1 (Prunus avium)；桑树：AIL23819.1 (Morus notabilis)；阿月浑子：XP_031267739.1 (Pistacia vera)；白杨树：XP_034917931.1 (Populus alba)；

Figure 5. Multiple sequence alignment of VfNHX1 homologous protein

Note: 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. VfNHX1 amino acid types and contents

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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 analysis (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. Structure prediction of VfNHX1 protein

A: Protein secondary structure prediction; B: Protein three-dimensional structure prediction

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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

用LSD法分别对叶和根进行多重比较，标有不同大写字母表示组内差异极显著（P＜0.01）,标有不同小写字母表示组内差异显著（P＜0.05）,标有相同小写字母表示组内差异不显著（P＞0.05）。

Figure 10. The relative expression level of VfNHX1 in leaves and roots under different salt stress time

Multiple comparisons of leaves and roots were performed separately by LSD method, labelled with different upper case letters indicate highly significant (P＜0.01), labelled with different lower case letters indicate significant (P＜0.05), and labelled with the same lower case letters indicate non-significant (P＞0.05) differences within the group.

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图 11 盐胁迫下酵母生长

A：稀释倍数。B：ns表示无显著差异(P＞0.05)，大写字母表示差异极显著（P＜0.01）,小写字母表示差异显著（P＜0.05）,相同小写字母表示差异不显著（P＞0.05）。

Figure 11. Growth of yeast under salt stress

A: Dilution factor.B: ns indicates no significant difference(P＞0.05), uppercase letters indicate highly significant differences (P＜0.01), lowercase letters indicate significant differences (P＜0.05), and the same lowercase letters indicate non-significant differences (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 gene cloning and fluorescence quantitative PCR

引物名称 Primername	引物序列(5′-3′) Primer sequence (5′→3′)	用途 Application
NHX1-127F	CTTGAGGAGAATCGGTGGATGAA	中间片段克隆
NHX1-1270R	GTGCTTGTGATCATGATTGCATTG	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
UPS	CTAATACGACTCACTATAGGGC	3′5′ first round primers
NHX1-438F	TGATGCTACCTCAGTGGTGCTT	荧光定量PCR
NHX1-622R	AGTGCCTGCCAATGTAGAGC	qRT-PCR
ELF1A-F	GTGAAGCCCGGTATGCTTGT	内参基因
ELF1A-R	CTTGAGATCCTTGACTGCAACATT	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/

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