秋茄KoWRKY43基因克隆、表达与生物信息学分析

doi:10.3969/j.issn.1004-1524.20231173

浙江农业学报 ›› 2024, Vol. 36 ›› Issue (8): 1832-1843.DOI: 10.3969/j.issn.1004-1524.20231173

秋茄KoWRKY43基因克隆、表达与生物信息学分析

蒋文骏¹(), 舒红锁², 陈正满³, 任典挺², 杨党¹^,⁴, 田荣江¹, 杜照奎¹^,⁴^,^*()

1.台州学院生命科学学院,浙江台州 318000
2.三门县自然资源和规划局,浙江三门 317100
3.台州循环经济发展有限公司,浙江台州 318000
4.台州学院浙江省植物进化生态学与保护重点实验室,浙江台州 318000

收稿日期:2023-10-09 出版日期:2024-08-25 发布日期:2024-09-06
作者简介:*杜照奎,E-mail: dzk@tzc.edu.cn
蒋文骏(2003—),男,浙江仙居人,本科生,主要从事植物分子生物学研究。E-mail: 3202250665@qq.com
通讯作者: 杜照奎
基金资助:
台州市科技计划(22nya05);浙江省自然资源厅科技项目(2021-43);浙江省基础公益研究计划(GN21C160013)

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

摘要/Abstract

摘要：

转录因子WRKY在开花植物中广泛存在,参与并调节植物的生长发育与防御反应等。为探究红树植物秋茄WRKY基因在非生物胁迫过程中的作用,以秋茄幼苗为试验材料,提取叶片总RNA,通过反转录PCR(RT-PCR)技术克隆获得KoWRKY43基因(GenBank登录号OR789874),采用生物信息学手段分析其基因序列和蛋白质结构特点,利用实时荧光定量PCR(qRT-PCR)技术研究其表达模式。结果表明,KoWRKY43基因开放阅读框(ORF)为942 bp,编码313个氨基酸,蛋白质分子式为C₁₄₈₄H₂₄₁₅N₄₃₉O₄₅₉S₁₄,分子量为34.2 ku,理论等电点为9.74,无信号肽,无跨膜结构,定位于细胞核,与木薯、银白杨和簸箕柳亲缘关系相对较近。qPCR检测显示,KoWRKY43基因在秋茄根中表达量最大,显著高于茎、叶、花和果实;幼叶KoWRKY43的表达量受NaCl诱导持续上升,在24 h表达量最大;激素水杨酸和脱落酸的诱导使其表达量先升高后降低,在6 h达到峰值;茉莉酸甲酯在24 h内均未显著改变其表达水平。研究结果为后续开展KoWRKY43基因的功能研究和培育抗逆秋茄品种提供了理论基础。

关键词: 秋茄, KoWRKY43基因, 生物信息学分析, 基因克隆, 激素

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

中图分类号:

S718.4

蒋文骏, 舒红锁, 陈正满, 任典挺, 杨党, 田荣江, 杜照奎. 秋茄KoWRKY43基因克隆、表达与生物信息学分析[J]. 浙江农业学报, 2024, 36(8): 1832-1843.

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.

图/表 14

表1 引物信息

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

图1 秋茄叶片总RNA电泳图泳道M,DNA marker DL 2 000;泳道1,提取的总RNA。

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

图2 KoWRKY43基因的PCR扩增泳道M,DNA marker DL 2 000;泳道1和2,KoWRKY43基因。

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

图3 KoWRKY43基因的cDNA序列和预测氨基酸序列红色和蓝色下划线分别代表KoWRKY43蛋白N端WRKYGQK和C端CX5CX23HXH保守序列。

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.

表2 KoWRKY43蛋白的氨基酸组成

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

图4 KoWRKY43蛋白磷酸化位点预测

Fig.4 Prediction of phosphorylation sites for KoWRKY43 protein

图5 KoWRKY43蛋白亲水性和疏水性预测

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

图6 KoWRKY43蛋白二级结构预测蓝色代表α螺旋;红色代表延伸链;绿色代表β转角;紫色代表无规卷曲。

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

图7 KoWRKY43蛋白三级结构预测

Fig.7 Tertiary structure prediction of KoWRKY43 protein

图8 秋茄KoWRKY43蛋白与其他植物WRKY蛋白的氨基酸序列比对

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

图9 秋茄KoWRKY43蛋白与其他植物WRKY蛋白的系统发育树

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

图10 秋茄KoWRKY43基因上游2 000 bp启动子区顺式作用元件预测 ARE,抗氧化反应元件;ABRE,ABA响应元件;MYB,MYB结合位点;AuxRR,生长素响应元件。

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.

图11 KoWRKY43基因在秋茄不同器官中的相对表达量叶中的相对表达量作为其他器官表达量的相对标准,定义为1。误差线表示标准误,不同小写字母表示在0.05水平上器官间差异显著。

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.

图12 NaCl、水杨酸、脱落酸和茉莉酸甲酯胁迫下秋茄叶片中KoWRKY43基因的表达量变化 A,200 mmol·L-1 NaCl胁迫;B,100 μmol·L-1水杨酸胁迫;C,100 μmol·L-1脱落酸胁迫;D,100 μmol·L-1茉莉酸甲酯胁迫。处理时间为0 h的表达量作为其他处理时间表达量的相对标准,定义为1;误差线表示标准误,柱上无相同小写字母表示在0.05水平上不同处理时间的表达量差异显著。

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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秋茄KoWRKY43基因克隆、表达与生物信息学分析

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

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