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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(X):1−11
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(X):1−11
JIN W H, FAN Y C, LI P, et al. Cloning and preliminary functional verification of VfNHX1 in faba bean (Vicia faba L.) [J]. Fujian Journal of Agricultural Sciences,2024,39(X):1−11
Citation: JIN W H, FAN Y C, LI P, et al. Cloning and preliminary functional verification of VfNHX1 in faba bean (Vicia faba L.) [J]. Fujian Journal of Agricultural Sciences,2024,39(X):1−11

蚕豆VfNHX1基因克隆及初步功能验证

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

Cloning and preliminary functional verification of VfNHX1 in faba bean (Vicia faba L.)

  • 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。
  • 图  1  蚕豆中的RNA

    Figure  1.  RNA in Vicia faba L.

    M: Marker, DL2000;1: RNA.

    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.

    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

    图  4  VfNHX1蛋白进化树

    自展值:Bootstrap value;

    Figure  4.  Phylogenetic tree of VfNHX1 protein

    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%;

    图  6  VfNHX1氨基酸种类及含量

    Figure  6.  VfNHX1 amino acid types and contents

    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

    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

    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

    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.

    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).

    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/
    下载: 导出CSV
  • [1] 罗达, 宋锋惠, 卢明艳, 等. 盐胁迫对平欧杂种榛根系生理生化特性的影响 [J]. 东北林业大学学报, 2024, 52(4):29−33,45. doi: 10.3969/j.issn.1000-5382.2024.04.005

    LUO D, SONG F H, LU M Y, et al. Effects of salt stress on the physiological and biochemical characteristics of ping’ou hybrid hazelnut root systems [J]. Journal of Northeast Forestry University, 2024, 52(4): 29−33,45. (in Chinese) doi: 10.3969/j.issn.1000-5382.2024.04.005
    [2] SHELKE D B, NIKALJE G C, CHAMBHARE M R, et al. Na+ and Cl− induce differential physiological, biochemical responses and metabolite modulations in vitro in contrasting salt-tolerant soybean genotypes [J]. 3 Biotech, 2019, 9(3): 91. doi: 10.1007/s13205-019-1599-6
    [3] 朱晨晨, 史昆, 何沁坤, 等. 混合盐碱胁迫对紫花苜蓿幼苗生理和基因表达的影响 [J]. 草地学报, 2024, 32(4):1044−1054.

    ZHU C C, SHI K, HE Q K, et al. Effects of mixed saline-alkali stress on physiology and gene expression of alfalfa seedlings [J]. Acta Agrestia Sinica, 2024, 32(4): 1044−1054. (in Chinese)
    [4] ZHANG H L, YU F F, XIE P, et al. A Gγ protein regulates alkaline sensitivity in crops [J]. Science, 2023, 379(6638): eade8416. doi: 10.1126/science.ade8416
    [5] KUMAR A, SINGH S, GAURAV A K, et al. Plant growth-promoting bacteria: Biological tools for the mitigation of salinity stress in plants [J]. Frontiers in Microbiology, 2020, 11: 1216. doi: 10.3389/fmicb.2020.01216
    [6] KARIM R, BOUCHRA B, FATIMA G, et al. Plant NHX antiporters: From function to biotechnological application, with case study [J]. Current Protein & Peptide Science, 2021, 22(1): 60−73.
    [7] 万玺宏, 张会龙, 朱建峰, 等. 液泡膜转运蛋白在植物耐盐性调控中的作用 [J]. 植物生理学报, 2024, 60(2):295−310.

    WAN X H, ZHANG H L, ZHU J F, et al. The role of tonoplast transporters in the regulation of salt tolerance in plants [J]. Plant Physiology Journal, 2024, 60(2): 295−310. (in Chinese)
    [8] 银芳柳, 毛晓菲, 曾幼玲. 盐生植物盐爪爪液泡膜钠氢反向运输载体基因(KfNHX1)遗传转化拟南芥的耐盐性鉴定 [J]. 新疆农业科学, 2021, 58(3):565−572.

    YIN F L, MAO X F, ZENG Y L. Salt-tolerant identification of genetic transformation in Arabidopsis with the KfNHX1 gene from the halophyte Kalidium foliatum [J]. Xinjiang Agricultural Sciences, 2021, 58(3): 565−572. (in Chinese)
    [9] 李晓薇, 郭嘉, 王鑫, 等. 羊草液泡膜Na+/H+逆向转运蛋白基因LcNHX1的克隆及功能分析 [J]. 中国草地学报, 2017, 39(5):1−9.

    LI X W, GUO J, WANG X, et al. Cloning and functional analysis of a vacuolar Na+/H+ antiporter gene LcNHX1 from Leymus chinensis [J]. Chinese Journal of Grassland, 2017, 39(5): 1−9. (in Chinese)
    [10] HUANG Y, ZHANG X X, LI Y H, et al. Overexpression of the Suaeda salsa SsNHX1 gene confers enhanced salt and drought tolerance to transgenic Zea mays [J]. Journal of Integrative Agriculture, 2018, 17(12): 2612−2623. doi: 10.1016/S2095-3119(18)61998-7
    [11] BIMURZAYEV N, SARI H, KURUNC A, et al. Effects of different salt sources and salinity levels on emergence and seedling growth of faba bean genotypes [J]. Scientific Reports, 2021, 11: 18198. doi: 10.1038/s41598-021-97810-6
    [12] NASRALLAH A K, ATIA M A M, ABD EL-MAKSOUD R M, et al. Salt priming as a smart approach to mitigate salt stress in faba bean (Vicia faba L. ) [J]. Plants, 2022, 11(12): 1610. doi: 10.3390/plants11121610
    [13] ÁLVAREZ-IGLESIAS L, PUIG C G, REVILLA P, et al. Faba bean as green manure for field weed control in maize [J]. Weed Research, 2018, 58(6): 437−449. doi: 10.1111/wre.12335
    [14] OUZOUNIDOU G, ILIAS I F, GIANNAKOULA A, et al. Effect of water stress and NaCl triggered changes on yield, physiology, biochemistry of broad bean (Vicia faba) plants and on quality of harvested pods [J]. Biologia, 2014, 69(8): 1010−1017. doi: 10.2478/s11756-014-0397-1
    [15] 陈江飞, 余津铭, 杨建坤, 等. 茶树Na+/H+逆向转运蛋白基因CsNHX1、CsNHX2的克隆及表达分析 [J]. 茶叶科学, 2018, 38(6):559−568. doi: 10.3969/j.issn.1000-369X.2018.06.002

    CHEN J F, YU J M, YANG J K, et al. Cloning and expression analysis of Na+/H+Antiporter gene CsNHX1 and CsNHX2 in tea plant(Camellia sinensis) [J]. Journal of Tea Science, 2018, 38(6): 559−568. (in Chinese) doi: 10.3969/j.issn.1000-369X.2018.06.002
    [16] 唐欣, 王瑞辉, 杨秀艳, 等. 唐古特白刺液泡膜Na+/H+逆向运输蛋白基因NtNHX1的克隆与表达分析 [J]. 林业科学, 2014, 50(3):38−44.

    TANG X, WANG R H, YANG X Y, et al. Isolation and expression analysis of a vacuolar membrane Na+/H+ antiporter gene NtNHX1 from Nitraria tangutorum [J]. Scientia Silvae Sinicae, 2014, 50(3): 38−44. (in Chinese)
    [17] 陈心仪, 吴成英, 贺海皓, 等. 滇水金凤4CL基因的克隆及表达分析 [J]. 福建农业学报, 2024, 39(1):40−48.

    CHEN X Y, WU C Y, HE H H, et al. Cloning and expression of 4CLs in Impatiens uliginosa [J]. Fujian Journal of Agricultural Sciences, 2024, 39(1): 40−48. (in Chinese)
    [18] 姚娜, 云岚, 艾芊, 等. 冬箭筈豌豆耐盐基因NHX1克隆及表达分析[J/OL]. 草地学报, 2024: 1-16. (2024-04-08). https://kns.cnki.net/kcms/detail/11.3362.S.20240405.1524.002.html.

    YAO N, YUN L, AI Q, et al. Cloning and expression analysis of salt-tolerant gene NHX1 in Vicia villosa Roth[J/OL]. Acta Agrestia Sinica, 2024: 1-16. (2024-04-08). https://kns.cnki.net/kcms/detail/11.3362.S.20240405.1524.002.html.(in Chinese)
    [19] 李霞, 孔丹宇, 刘传鑫, 等. 滨豇豆VmNHX基因克隆与表达分析 [J]. 分子植物育种, 2024, 22(2):402−413.

    LI X, KONG D Y, LIU C X, et al. Cloning and expression analysis of VmNHX gene in Vigna marina [J]. Molecular Plant Breeding, 2024, 22(2): 402−413. (in Chinese)
    [20] 董禄禄, 秦晓春, 党振华. 长叶红砂液泡膜Na+/H+逆向转运蛋白基因的克隆及表达特性 [J]. 西北植物学报, 2015, 35(11):2164−2170. doi: 10.7606/j.issn.1000-4025.2015.11.2164

    DONG L L, QIN X C, DANG Z H. Isolation and expression of vacuolar membrane Na+/H+ antiporter gene in reaumuriatrigyna [J]. Acta Botanica Boreali-Occidentalia Sinica, 2015, 35(11): 2164−2170. (in Chinese) doi: 10.7606/j.issn.1000-4025.2015.11.2164
    [21] 张永利, 孟晓烨, 孙婷梅, 等. 珠美海棠Mz2NHX1基因的克隆和序列分析 [J]. 江苏农业科学, 2015, 43(9):20−25.

    ZHANG Y L, MENG X Y, SUN T M, et al. Cloning and sequence analysis of Mz2NHX1 gene from Malus jumeiensis [J]. Jiangsu Agricultural Sciences, 2015, 43(9): 20−25. (in Chinese)
    [22] 邱全胜. 拟南芥NHX5和NHX6: 离子平衡与蛋白质运输 [J]. 中国科学: 生命科学, 2017, 47(8):839−846. doi: 10.1360/N052016-00351

    QIU Q S. Arabidopsis NHX5 and NHX6: Ion homeostasis and protein transport [J]. Scientia Sinica (Vitae), 2017, 47(8): 839−846. (in Chinese) doi: 10.1360/N052016-00351
    [23] 李源, 蔡勤安, 马瑞, 等. 植物Na+/H+逆向转运蛋白研究进展 [J]. 山东农业科学, 2022, 54(10):143−152.

    LI Y, CAI Q A, MA R, et al. Research progress of plant Na+/H+ antiporter [J]. Shandong Agricultural Sciences, 2022, 54(10): 143−152. (in Chinese)
    [24] 边晨凯, 龙定沛, 刘雪琴, 等. 桑树Na+/H+逆向转运蛋白基因(MnNHX1)的克隆与耐盐力表达 [J]. 林业科学, 2015, 51(8):16−25.

    BIAN C K, LONG D P, LIU X Q, et al. Cloning and expression to salt stress of Na+/H+ antiporter gene(MnNHX1) in mulberry tree [J]. Scientia Silvae Sinicae, 2015, 51(8): 16−25. (in Chinese)
    [25] 许浩宇, 赵颖, 阮倩, 等. 不同混合盐碱下藜麦幼苗的抗性研究 [J]. 草业学报, 2023, 32(1):122−130. doi: 10.11686/cyxb2021500

    XU H Y, ZHAO Y, RUAN Q, et al. Resistance of quinoa seedlings under different salt-alkali stress levels [J]. Acta Prataculturae Sinica, 2023, 32(1): 122−130. (in Chinese) doi: 10.11686/cyxb2021500
    [26] 李宁宁, 孙亚卿, 李国龙. 高糖甜菜BvNHX1基因的克隆及表达特性分析 [J]. 分子植物育种, 2021, 19(16):5250−5257.

    LI N N, SUN Y Q, LI G L. Cloning and expression analysis of BvNHX1 from beta vulgaris with high sucrose [J]. Molecular Plant Breeding, 2021, 19(16): 5250−5257. (in Chinese)
    [27] 高玉龙, 宋中邦, 李梅云, 等. 烟草NtNHX1-3基因的克隆及表达特性 [J]. 西北植物学报, 2018, 38(12):2201−2206. doi: 10.7606/j.issn.1000-4025.2018.12.2201

    GAO Y L, SONG Z B, LI M Y, et al. Cloning and expression characteristics of tobacco NtNHX1-3Gene [J]. Acta Botanica Boreali-Occidentalia Sinica, 2018, 38(12): 2201−2206. (in Chinese) doi: 10.7606/j.issn.1000-4025.2018.12.2201
    [28] 李玥, 肖如雪, 芮蕊, 等. 筇竹Na+/H+逆向转运蛋白基因克隆与表达分析 [J]. 分子植物育种, 2021, 19(10):3235−3242.

    LI Y, XIAO R X, RUI R, et al. Cloning and expression analysis on QtNHX1 gene from Qiongzhuea tumidinoda [J]. Molecular Plant Breeding, 2021, 19(10): 3235−3242. (in Chinese)
    [29] 喻珊, 胡艳平, 丛心黎, 等. 海马齿Na+/H+逆转运蛋白基因SpNHX1的克隆及表达模式 [J]. 热带生物学报, 2015, 6(2):127−133. doi: 10.3969/j.issn.1674-7054.2015.02.004

    YU S, HU Y P, CONG X L, et al. Isolation and expression analysis of Na+/H+ antiporter gene SpNHX1 from Sesuvium portulacastrumand L [J]. Journal of Tropical Biology, 2015, 6(2): 127−133. (in Chinese) doi: 10.3969/j.issn.1674-7054.2015.02.004
    [30] 王立光, 陈军, 叶春雷, 等. 酿酒酵母BJ3505 NHX1基因突变株的构建及功能验证 [J]. 生物技术通报, 2018, 34(12):152−158.

    WANG L G, CHEN J, YE C L, et al. Mutant construction and functional validation of NHX1 in Saccharomyces cerevisiae BJ3505 [J]. Biotechnology Bulletin, 2018, 34(12): 152−158. (in Chinese)
    [31] 刘威, 李慧, 蔺经, 等. 杜梨PbNHX1基因的克隆、表达分析及功能验证 [J]. 果树学报, 2018, 35(2):137−146.

    LIU W, LI H, LIN J, et al. Cloning, expression and functional analysis of PbNHX1 gene in Pyrus betulaefolia [J]. Journal of Fruit Science, 2018, 35(2): 137−146. (in Chinese)
    [32] 赵云霞, 郭丹丽, 魏艳玲, 等. 新疆无苞芥Na+/H+逆向转运蛋白基因OpNHX1的克隆、表达分析与功能验证 [J]. 生物技术通报, 2014, (7):74−80.

    ZHAO Y X, GUO D L, WEI Y L, et al. Cloning, expressing and functional analysis of Na+/H+ antiporter gene OpNHX1 from Olimarabidopsis pumila in Xinjiang [J]. Biotechnology Bulletin, 2014(7): 74−80. (in Chinese)
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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

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