Cloning, expression, and bioinformatics analysis of KoWRKY43 gene in Kandelia obovata

doi:10.3969/j.issn.1004-1524.20231173

Acta Agriculturae Zhejiangensis ›› 2024, Vol. 36 ›› Issue (8): 1832-1843.DOI: 10.3969/j.issn.1004-1524.20231173

• Horticultural Science • Previous Articles Next Articles

Cloning, expression, and bioinformatics analysis of KoWRKY43 gene in Kandelia obovata

JIANG Wenjun¹(), SHU Hongsuo², CHEN Zhengman³, REN Dianting², YANG Dang¹^,⁴, TIAN Rongjiang¹, DU Zhaokui¹^,⁴^,^*()

1. School of Life Sciences, Taizhou University, Taizhou 318000, Zhejiang, China
2. Sanmen Bureau of Natural Resources and Planning, Sanmen 317100, Zhejiang, China
3. Taizhou Circular Economy Development Co., Ltd., Taizhou 318000, Zhejiang, China
4. Zhejiang Provincial Key Laboratory of Plant Evolutionary Ecology and Conservation, Taizhou University, Taizhou 318000, Zhejiang, China

Received:2023-10-09 Online:2024-08-25 Published:2024-09-06
Contact: DU Zhaokui

Abstract

Abstract:

The transcription factor WRKY is widespread in flowering plants, and regulates plant growth, development and defense responses. To explore the role of the WRKY gene in Kandelia obovata under abiotic stresses, total RNA was extracted from the leaves of K. obovata, and then the KoWRKY43 gene (GenBank accession number OR789874) was cloned by reverse transcription-PCR (RT-PCR); the gene sequence and protein structure were analyzed by bioinformatics, and its expression pattern was studied by real-time fluorogenic quantitative PCR (qRT-PCR). The results showed that the gene contained an open reading frame (ORF) of 942 bp, encoding 313 amino acids. The protein KoWRKY43, with a molecular formula of C₁₄₈₄H₂₄₁₅N₄₃₉O₄₅₉S₁₄, a molecular weight of 34.2 ku, and a theoretical isoelectric point of 9.74, had no signal peptide or transmembrane structure, and it was predicted to be located in the nucleus. Phylogenetic analysis indicated that KoWRKY43 had the most homology with Manihot esculenta, Populus alba and Salix suchowensis. The qRT-PCR analysis showed that the expression abundance of KoWRKY43 gene was highest in roots, which was significantly higher than that in stems, leaves, flowers and fruits. The expression level of the KoWRKY43 in young leaves was induced by NaCl, salicylic acid (SA) and abscisic acid (ABA), and the highest expression levels were observed at 24 h, 6 h and 6 h, respectively. However, methyl jasmonate (MeJA) did not significantly alter the expression level of KoWRKY43 within 24 h. The present research provided a theoretical basis for subsequent functional studies of the gene and the cultivation of stress-resistant K. obovata varieties.

Key words: Kandelia obovata, KoWRKY43 gene, bioinformatics analysis, gene cloning, hormone

CLC Number:

S718.4

JIANG Wenjun, SHU Hongsuo, CHEN Zhengman, REN Dianting, YANG Dang, TIAN Rongjiang, DU Zhaokui. Cloning, expression, and bioinformatics analysis of KoWRKY43 gene in Kandelia obovata[J]. Acta Agriculturae Zhejiangensis, 2024, 36(8): 1832-1843.

Figures/Tables 14

Table 1 Information of primers

基因 Gene	上游引物 Forward primer(5'→3')	下游引物 Reverse primer(5'→3')	用途 Purpose
KoWRKY43	ATGGATGAGACCACGCCGGA	CTAAAATATCTCATGATAAC	基因扩增Gene amplification
KoWRKY43	CACCGATCCAGCAGATTCCC	ACAAGAAGGACGAAAGCGGA	实时荧光定量PCR Real-time fluorogenic quantitative PCR
18S rRNA	GGGGCTCGAAGACGATCAGA	TTAAGCCGCAGGCTCCACTC	内参基因Internal reference gene

Fig.1 Electrophoresis image of total RNA from Kandelia obovata leaf Lane M, DNA marker DL 2 000; Lane 1, Total RNA.

Fig.2 The PCR result of KoWRKY43 gene Lane M, DNA marker DL 2 000; Lane 1 and 2, KoWRKY43 gene.

Fig.3 Coding sequence and predicted amino acid sequence of KoWRKY43 gene Red and blue underlines represent the N-terminal WRKYGQK and C-terminal CX5CX23HXH conserved sequences of KoWRKY43 protein, respectively.

Table 2 Amino acid composition of KoWRKY43 protein

氨基酸种类 Type of amino acid	数量 Quantity	占氨基酸总量的百分比 Percent of total amino acids/%	氨基酸的种类 Type of amino acid	数量 Quantity	占氨基酸总量的百分比 Percent of total amino acids/%
丙氨酸Alanine	25	8.00	亮氨酸Leucine	22	7.00
精氨酸Arginine	20	6.40	赖氨酸Lysine	26	8.30
天冬氨酰Asparagine	15	4.80	甲硫氨酸Methionine	7	2.20
天冬氨酸Aspartic acid	15	4.80	苯丙氨酸Phenylalanine	8	2.60
半胱氨酸Cysteine	7	2.20	脯氨酸Proline	22	7.00
谷氨酰胺Glutamine	12	3.80	丝氨酸Serine	43	13.70
谷氨酸Glutamic acid	12	3.80	苏氨酸Threonine	13	4.20
甘氨酸Glycine	15	.80	色氨酸Tryptophan	1	0.30
组氨酸Histidine	6	1.90	酪氨酸Tyrosine	8	2.60
异亮氨酸Isoleucine	13	4.20	缬氨酸Valine	23	7.30

Fig.4 Prediction of phosphorylation sites for KoWRKY43 protein

Fig.5 Prediction of hydrophilic and hydrophobic properties of KoWRKY43 protein

Fig.6 Secondary structure prediction of KoWRKY43 protein Blue, α-Helix; Red, Extended chain; Green, β-Turn; Purple, Random coil.

Fig.7 Tertiary structure prediction of KoWRKY43 protein

Fig.8 Amino acid sequence alignment of Kandelia obovata KoWRKY43 protein with other plant WRKY proteins

Fig.9 Phylogenetic tree of Kandelia obovata KoWRKY43 protein and other plant WRKY proteins

Fig.10 Prediction of cis-acting elements in the upstream 2 000 bp promoter region of Kandelia obovata KoWRKY43 gene ARE, Antioxidant responsive element; ABRE, ABA-responsive element; MBS, MYB binding site; AuxRR, Auxin-responsive element.

Fig.11 Relative expression levels of KoWRKY43 gene in different organs of Kandelia obovata The expression level in leaf is defined as a relative standard for the expression of other organs, with error bars indicating standard error, and different lowercase letters indicate significant differences among organs at the 0.05 level.

Fig.12 Expression changes of KoWRKY43 gene in the leaf of Kandelia obovata under stress conditions of NaCl, salicylic acid, abscisic acid, and methyl jasmonate A, 200 mmol·L-1 NaCl stress; B, 100 μmol·L-1 salicylic acid stress; C, 100 μmol·L-1 abscisic acid stress; D, 100 μmol·L-1 methyl jasmonate stress. The expression level at 0 h is defined as a relative standard for other treatment times, defined as 1, error bars represent standard errors, and bars without the same lowercase letters indicate significant differences in expression levels at different treatment times at the 0.05 level.

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Cloning, expression, and bioinformatics analysis of KoWRKY43 gene in Kandelia obovata

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