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

doi:10.3969/j.issn.1004-1524.2022.04.12

Acta Agriculturae Zhejiangensis ›› 2022, Vol. 34 ›› Issue (4): 756-765.DOI: 10.3969/j.issn.1004-1524.2022.04.12

• Horticultural Science • Previous Articles Next Articles

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

Abstract

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

CLC Number:

S529

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.

Figures/Tables 12

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

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).

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.

Fig.3 Amino acid sequence of VfHKT1

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

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

Fig.5 Signal peptide prediction for VfHKT1;1

Fig.6 Transmembrane domain prediction of VfHKT1;1

Fig.7 CDD analysis of VfHKT1;1

Fig.8 The secondary structure prediction of VfHKT1;1

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

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.

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

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