蚕豆耐盐相关基因VfHKT1;1的克隆、生物信息学分析及表达特性

doi:10.3969/j.issn.1004-1524.2022.04.12

浙江农业学报 ›› 2022, Vol. 34 ›› Issue (4): 756-765.DOI: 10.3969/j.issn.1004-1524.2022.04.12

蚕豆耐盐相关基因VfHKT1;1的克隆、生物信息学分析及表达特性

樊有存¹(), 张红岩¹, 杨旭升², 韩芊², 刘玉皎¹, 武学霞¹^,^*()

1.青海大学农林科学院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 810000
2.青海大学生态环境工程学院,青海西宁810000

收稿日期:2020-11-24 出版日期:2022-04-25 发布日期:2022-04-28
通讯作者: 武学霞
作者简介:*武学霞,E-mail: xuexun111@163.com
樊有存(1994—),女,青海西宁人,硕士研究生,从事蚕豆种质创新与改良利用研究。E-mail: 2685375107@qq.com
基金资助:
青海省自然科学基金(2018-ZJ-940Q);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自主课题(2017-ZZ-12);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自主课题(2016-ZZ-04);国家食用豆产业技术体系建设专项(CARS-08)

Cloning, bioinformatics analysis and gene expression pattern of VfHKT1; 1 in Vicia faba L.

FAN Youcun¹(), ZHANG Hongyan¹, YANG Xusheng², HAN Qian², LIU Yujiao¹, WU Xuexia¹^,^*()

1. State Key Laboratory of Plateau Ecology and Agriculture, Academy of Agricultural and Forestry, Qinghai University, Xining 810000, China
2. College of Eco-Environmental Engineering, Qinghai University, Xining 810000, China

Received:2020-11-24 Online:2022-04-25 Published:2022-04-28
Contact: WU Xuexia

摘要/Abstract

摘要：

高亲和性钾离子转运蛋白(high affinity K⁺ transporter, HKT)能够调节细胞及整株的Na⁺/K⁺转运,在植物盐胁迫调控中发挥着重要作用。采用RT-PCR和PCR方法克隆获得蚕豆VfHKT1;1基因全长编码序列,该基因包含1 659 bp的开放阅读框,编码552个氨基酸。系统进化树分析发现,该蛋白属于HKT第Ⅰ亚家族,命名为VfHKT1;1,且VfHKT1;1蛋白氨基酸序列与苜蓿MtHKT1;1的序列相似度最高。生物信息学分析发现,VfHKT1;1蛋白结构稳定,不具有信号肽结构,含有8个跨膜区域,且蛋白含有HKT家族典型的保守结构域TrKH,属于典型的跨膜离子转运体。实时荧光定量PCR分析表明,VfHKT1;1基因的表达具有明显的组织特异性。在盐胁迫诱导下,VfHKT1;1基因在根中的表达水平受到抑制,而在叶片中的表达水平上升,且在盐处理1h后达到最高值,随后随着盐处理时间的延长又逐渐下降。

关键词: 蚕豆, 基因克隆, 生物信息学分析, 表达特性

Abstract:

High affinity K⁺ transporter (HKT) involve in salt tolerance response through maintaining Na⁺/K⁺ homeostasis in cellar and whole plant. VfHKT1;1 gene was cloned using RT-PCR and PCR in this work and the gene encoded an open reading frame (1 659 bp) consisting of 552 amino acids. Phylogenetic tree analysis indicated that VfHKT1;1 belong to HKT subfamily Ⅰ and shared the highest sequence similarity with MtHKT1;1, so it was named as VfHKT1;1. The bioinformatics analysis showed that VfHKT1;1 was stable, which also contained 8 transmembrane regions and no signal peptide. It was supposed to be an ion transporter which usually located at membrane because it contains a typical TrKH conserved functional domain of HKT family. Real-time fluorescent quantitative PCR showed different expression levels of VfHKT1;1 gene in root and leaf. The expression of VfHKT1;1 gene in root was suppressed under salt stress.Whereas it had higher transcript levels in leaf under salt stress, the expression peak appeared at 1 h after NaCl treatment. Then, the expression level of VfHKT1;1 gene gradually declined with the NaCl treatment time.

Key words: Vicia faba L., gene cloning, bioinformatics analysis, gene expression

中图分类号:

S529

樊有存, 张红岩, 杨旭升, 韩芊, 刘玉皎, 武学霞. 蚕豆耐盐相关基因VfHKT1;1的克隆、生物信息学分析及表达特性[J]. 浙江农业学报, 2022, 34(4): 756-765.

FAN Youcun, ZHANG Hongyan, YANG Xusheng, HAN Qian, LIU Yujiao, WU Xuexia. Cloning, bioinformatics analysis and gene expression pattern of VfHKT1; 1 in Vicia faba L.[J]. Acta Agriculturae Zhejiangensis, 2022, 34(4): 756-765.

图/表 12

表1 qRT-PCR引物序列

Table 1 The qRT-PCR primer sequences

引物名称 Primer name	引物序列 Primer sequence(5'-3')	产物大小 Product size/bp
ELF1A-F	GTGAAGCCCGGTATGCTTGT	169
ELF1A-R	GATGCTGAGGATATTCAACCCC
HKT1-1050F	TGGGTTGCTTGTGGTTCAAG	176
HKT1-1232R	ACAAACAACACCAACACGGC
HKT1-889F	GGGAACACGTTGTACCCTCC	158
HKT1-1069R	CTTGAACCACAAGCAACCCA
HKT1-265 F	TCAGCCACAACAGTTTCAAGC	159
HKT1-452 R	GCAAGACGAGCATGAGACGA
HKT1-877 F	CAAGTGCTTCTTGGGAACACG	183
HKT1-1057 R	GCAACCCAAAAACAGTAGCCA

图1 不同浓度NaCl处理7 d后的蚕豆幼苗 A,不同浓度盐处理下的株高(标尺=3 cm);B,不同浓度盐处理下的叶片表型(标尺=2 cm)。

Fig.1 Seedlings of faba bean irrigated with different concentration of NaCl for 7 days A,Plant height of faba bean under different NaCl concentrations(bar=3 cm);B,Leaf area of faba bean under different NaCl concentrations(bar=2 cm).

图2 VfHKT1基因扩增及鉴定 A,VfHKT1基因扩增;B,VfHKT1基因阳性克隆菌落鉴定。1、2、3和4均为PCR产物。

Fig.2 Cloning of VfHKT1 gene and identification of the positive clone A,Cloning of VfHKT1 gene;B,Identification of positive colonies of VfHKT1 gene. 1, 2, 3 and 4 represented PCR products.

图3 VfHKT1氨基酸序列

Fig.3 Amino acid sequence of VfHKT1

图4 VfHKT1;1与其他高等植物HKT蛋白的进化分析星号标记的为蚕豆VfHKT1;1 蛋白。

Fig.4 Phylogenetic analysis of VfHKT1;1 with other HKT transporters from higher plants Asterisk indicated the VfHKT1;1 of faba bean.

表2 VfHKT1;1蛋白氨基酸组成

Table 2 The amino acid composition of VfHKT1;1

氨基酸 Amino acid	数量 Quantity	百分比 Percentage/%	氨基酸 Amino acid	数量 Quantity	百分比 Percentage/%
丙氨酸Ala(A)	13	2.4	精氨酸Arg(R)	13	2.4
天冬酰胺Asn(N)	30	5.4	天冬氨酸Asp(D)	11	2.0
半胱氨酸Cys(C)	12	2.2	谷氨酰胺Gln(Q)	15	2.7
谷氨酸Glu(E)	26	4.7	甘氨酸Gly(G)	30	5.4
组氨酸His(H)	16	2.9	异亮氨酸Ile(I)	46	8.3
亮氨酸Leu(L)	80	14.5	赖氨酸Lys(K)	42	7.6
蛋氨酸Met(M)	17	3.1	苯丙氨酸Phe(F)	38	6.9
脯氨酸Pro(P)	15	2.7	丝氨酸Ser(S)	54	9.8
苏氨酸Thr(T)	32	5.8	色氨酸Trp(W)	6	1.1
酪氨酸Tyr(Y)	18	3.3	缬氨酸Val(V)	38	6.9
吡咯赖氨酸Pyl(0)	0	0	硒半胱氨酸Sec(U)	0	0

图5 VfHKT1;1信号肽预测

Fig.5 Signal peptide prediction for VfHKT1;1

图6 VfHKT1;1跨膜结构预测

Fig.6 Transmembrane domain prediction of VfHKT1;1

图7 VfHKT1;1保守结构域(CDD)分析

Fig.7 CDD analysis of VfHKT1;1

图8 VfHKT1;1二级结构预测

Fig.8 The secondary structure prediction of VfHKT1;1

图9 VfHKT1;1三级结构预测

Fig.9 Three-dimensional structure predictions of VfHKT1;1

图10 蚕豆VfHKT1;1基因表达分析 A,正常生长条件下VfHKT1;1基因在蚕豆根与叶片中的表达水平;B,NaCl处理下VfHKT1;1基因在蚕豆叶片中的表达水平;C,NaCl处理下VfHKT1;1基因在蚕豆根中的表达水平。图中所显示数值为平均数±标准差(n=3),*,**分别表示在P<0.05和P<0.01水平上差异显著。

Fig.10 Relative expression level of VfHKT1;1 gene in faba bean A,The expression of VfHKT1;1 gene in faba bean root and leaf under normal conditions;B,The expression of VfHKT1;1 gene in faba bean leaf under NaCl stress;C,The expression of VfHKT1;1 gene in faba bean root under NaCl stress. Values were means±SD (n=3). *,** represented significant differences at P<0.05 and P<0.01,respectively.

参考文献 31

[1]	王彦龙, 施建军, 盛丽, 等. 柴达木盆地重度盐碱地燕麦引种适应性评价[J]. 青海畜牧兽医杂志, 2019, 49(1): 36-41.
	WANG Y L, SHI J J, SHENG L, et al. Adaptive evaluation on oat varieties on severe saline-alkali land in Qaidam basin[J]. Chinese Qinghai Journal of Animal and Veterinary Sciences, 2019, 49(1): 36-41. (in Chinese with English abstract)
[2]	赵娜, 缪亚梅, 陈满峰, 等. 蚕豆耐盐性的研究进展[J]. 安徽农业科学, 2015, 43(18): 20-21.
	ZHAO N, MIAO Y M, CHEN M F, et al. Research progress on salt tolerance of faba bean[J]. Journal of Anhui Agricultural Sciences, 2015, 43(18): 20-21. (in Chinese with English abstract)
[3]	李爱萍, 郑开斌, 林碧英, 等. 蚕豆提高土壤肥力及土壤效力研究[J]. 农业与技术, 2007, 27(2): 61-63.
	LI A P, ZHENG K B, LIN B Y, et al. Study about how the broad bean improves the soil fertility and soil efficacy[J]. Agriculture & Technology, 2007, 27(2): 61-63. (in Chinese with English abstract)
[4]	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 URL
[5]	袁秀梅, 耿赛男, 郑梦圆, 等. 蚕豆根分泌物对紫色土有效养分及微生物数量的影响[J]. 中国生态农业学报, 2016, 24(7): 910-917.
	YUAN X M, GENG S N, ZHENG M Y, et al. Effects of faba bean(Vicia faba L.) root exudate on soil available nutrients and microbial population in different purple soils[J]. Chinese Journal of Eco-Agriculture, 2016, 24(7): 910-917. (in Chinese with English abstract)
[6]	叶旭君, 汪成宏, 王仁第, 等. 浙东沿海台灾受咸地冬季作物的适应性[J]. 浙江农业学报, 1998, 10(6): 298-301.
	YE X J, WANG C H, WANG R D, et al. Winter crop suitability in saline-alkali fields caused by typhoon-driven sea floods in eastern coastal area of Zhejiang Province[J]. Acta Agriculturae Zhejiangensis, 1998, 10(6): 298-301. (in Chinese with English abstract)
[7]	陈华涛, 陈新, 顾和平, 等. 大豆GmHKT6;2基因的克隆与表达特性分析[J]. 华北农学报, 2012, 27(3): 1-5.
	CHEN H T, CHEN X, GU H P, et al. Cloning and expression pattern analysis of GmHKT6;2in soybean[J]. Acta Agriculturae Boreali-Sinica, 2012, 27(3): 1-5. (in Chinese with English abstract)
[8]	陆潭, 陈华涛, 沈振国, 等. 植物钾通道与钾转运体研究进展[J]. 华北农学报, 2019, 34(S1): 372-379.
	LU T, CHEN H T, SHEN Z G, et al. Advance of potassium channels and transporters in plant[J]. Acta Agriculturae Boreali-Sinica, 2019, 34(S1): 372-379. (in Chinese with English abstract)
[9]	PLATTEN J D, COTSAFTIS O, BERTHOMIEU P, et al. Nomenclature for HKT transporters,key determinants of plant salinity tolerance[J]. Trends in Plant Science, 2006, 11(8):372-374. DOI URL
[10]	HORIE T, HAUSER F, SCHROEDER J I. HKT transporter-mediated salinity resistance mechanisms in Arabidopsis and monocot crop plants[J]. Trends in Plant Science, 2009, 14(12): 660-668. DOI URL
[11]	MÄSER P, HOSOO Y, GOSHIMA S, et al. Glycine residues in potassium channel-like selectivity filters determine potassium selectivity in four-loop-per-subunit HKT transporters from plants[J]. Proceedings of the National Academy of Sciences of the United States of America, 2002, 99(9): 6428-6433.
[12]	REHMAN H M, NAWAZ M A, SHAH Z H, et al. In-depth genomic and transcriptomic analysis of five K⁺ transporter gene families in soybean confirm their differential expression for nodulation[J]. Frontiers in Plant Science, 2017, 8: 804. DOI URL
[13]	CHEN H T, HE H, YU D Y. Overexpression of a novel soybean gene modulating Na⁺ and K⁺ transport enhances salt tolerance in transgenic tobacco plants[J]. PhysiologiaPlantarum, 2011, 141(1): 11-18.
[14]	CHEN H T, CHEN X, GU H P, et al. GmHKT1;4, a novel soybean gene regulating Na⁺/K⁺ ratio in roots enhances salt tolerance in transgenic plants[J]. Plant Growth Regulation, 2014, 73(3): 299-308. DOI URL
[15]	LI P, HOU W W, LIU Y J. Proteomic analysis of drought stress response on drought resistance for Vicia faba L. variety ‘Qinghai 13’ in Qinghai Plateau of China[J]. ActaAgronomicaSinica, 2019, 45(2): 267.
[16]	GUTIERREZ L, MAURIAT M, PELLOUX J, et al. Towards a systematic validation of references in real-time RT-PCR[J]. The Plant Cell, 2008, 20(7): 1734-1735. DOI URL
[17]	LIU W, SCHACHTMAN D P, ZHANG W. Partial deletion of a loop region in the high affinity K⁺ transporter HKT1 changes ionic permeability leading to increased salt tolerance[J]. The Journal of Biological Chemistry, 2000, 275(36): 27924-27932. DOI URL
[18]	HAUSER F, HORIE T. A conserved primary salt tolerance mechanism mediated by HKT transporters: a mechanism for sodium exclusion and maintenance of high K⁺/Na⁺ ratio in leaves during salinity stress[J]. Plant, Cell & Environment, 2010, 33(4): 552-565.
[19]	李旭娟, 刘洪博, 林秀琴, 等. 甘蔗KNOX基因(Sckn 1)的电子克隆及生物信息学分析[J]. 基因组学与应用生物学, 2015, 34(1): 136-142.
	LI X J, LIU H B, LIN X Q, et al. In silico cloning and bioinformatics analysis of KNOX gene in sugarcane(Sckn1)[J]. Genomics and Applied Biology, 2015, 34(1): 136-142. (in Chinese with English abstract)
[20]	ALI Z, PARK H C, ALI A, et al. TsHKT1;2, a HKT1 homolog from the extremophile Arabidopsis relative Thellungiella salsuginea, shows K⁺ specificity in the presence of NaCl[J]. Plant Physiology, 2012, 158(3): 1463-1474. DOI URL
[21]	KHAN M A, ALGHAMDI S S, AMMAR M H, et al. Transcriptome profiling of faba bean (Vicia faba L.) drought-tolerant variety Hassawi-2 under drought stress using RNA sequencing[J]. Electronic Journal of Biotechnology, 2019, 39: 15-29. DOI URL
[22]	WU X X, FAN Y C, LI L P, et al. The influence of soil drought stress on the leaf transcriptome of faba bean (Vicia faba L.) in the Qinghai-Tibet Plateau[J]. Biotech, 2020, 10(9): 1-16. DOI URL
[23]	DURELL S R, HAO Y L, NAKAMURA T, et al. Evolutionary relationship between K⁺ channels and symporters[J]. Biophysical Journal, 1999, 77(2): 775-788. DOI URL
[24]	VIEIRA-PIRES R S, SZOLLOSI A, MORAIS-CABRAL J H. The structure of the KtrAB potassium transporter[J]. Nature, 2013, 496(7445): 323-328. DOI URL
[25]	王甜甜, 郝怀庆, 冯雪, 等. 植物HKT蛋白耐盐机制研究进展[J]. 植物学报, 2018, 53(5):710-725. DOI
	WANG T T, HAO H Q, FENG X, et al. Research advances in the function of the high-affinity K⁺ transporter (HKT) proteins and plant salt tolerance[J]. Chinese Bulletin of Botany, 2018, 53(5):710-725. (in Chinese with English abstract)
[26]	MØLLER I S, GILLIHAM M, JHA D, et al. Shoot Na⁺ exclusion and increased salinity tolerance engineered by cell type-specific alteration of Na⁺ transport in Arabidopsis[J]. The Plant Cell, 2009, 21(7): 2163-2178. DOI URL
[27]	SUZUKI K, YAMAJI N, COSTA A, et al. OsHKT1;4-mediated Na⁺ transport in stems contributes to Na⁺ exclusion from leaf blades of rice at the reproductive growth stage upon salt stress[J]. BMC Plant Biology, 2016, 16: 22. DOI URL
[28]	KOBAYASHI N I, YAMAJI N, YAMAMOTO H, et al. OsHKT1;5 mediates Na⁺ exclusion in the vasculature to protect leaf blades and reproductive tissues from salt toxicity in rice[J]. The Plant Journal, 2017, 91(4): 657-670. DOI URL
[29]	WANG H, ZHANG M S, GUO R, et al. Effects of salt stress on ion balance and nitrogen metabolism of old and young leaves in rice (Oryza sativa L.)[J]. BMC Plant Biology, 2012, 12: 194. DOI URL
[30]	LAURIE S, FEENEY K A, MAATHUIS F J M, et al. A role for HKT1 in sodium uptake by wheat roots[J]. The Plant Journal, 2002, 32(2): 139-149. DOI URL
[31]	CAMPBELL M T, BANDILLO N, AL SHIBLAWI F R A, et al. Allelic variants of OsHKT1;1 underlie the divergence between indica and japonica subspecies of rice (Oryza sativa) for root sodium content[J]. PLoS Genetics, 2017, 13(6): e1006823. DOI URL

蚕豆耐盐相关基因VfHKT1;1的克隆、生物信息学分析及表达特性

Cloning, bioinformatics analysis and gene expression pattern of VfHKT1; 1 in Vicia faba L.

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[15]	梁敏华, 杨震峰, 苏新国, 宋春波. 桃果实八氢番茄红素合成酶基因的克隆与表达分析[J]. 浙江农业学报, 2018, 30(3): 399-405.