Cloning and preliminary functional verification of <i>VfNHX1</i> in faba bean (<i>Vicia faba</i> L.)

JIN Wenhai; FAN Youcun; LI Ping; FAN Huiling; HOU Wanwei; TENG Changcai; LIU Yujiao; WU Xuexia

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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

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

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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

Received Date: 2024-01-03
Accepted Date: 2024-06-02
Rev Recd Date: 2024-05-28

Available Online: 2024-07-10

Abstract

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

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References(32)

References

[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)