谷子SiMAPK3基因的克隆和表达特性分析

doi:10.13560/j.cnki.biotech.bull.1985.2022-0819

摘要/Abstract

摘要：

丝裂原激活的蛋白激酶（MAPK）是一类丝氨酸/苏氨酸蛋白质激酶，在植物生长发育、响应逆境胁迫及激素信号转导等方面具有重要作用。以谷子品种豫谷1号为试材，克隆与拟南芥AtMAPK3同源性最高的谷子SiMAPK3基因，并系统利用生物信息学方法分析SiMAPK3蛋白理化性质、结构与功能；利用RT-qPCR技术检测SiMAPK3基因在谷子拔节前期不同组织和不同非生物逆境胁迫下的表达水平。结果表明，谷子SiMAPK3基因开放阅读框为1 128 bp，编码一个含有376个氨基酸的蛋白，预测蛋白分子量为43 427.85 Da，等电点为5.46。谷子SiMAPK3为不含信号肽的亲水性膜外蛋白，其二级结构包含44.27%的α螺旋、14.67%的β折叠、5.07%的延伸链及36.00%的无规则卷曲，其第44-328位氨基酸之间含有Pkinase保守结构域，属于MAPK蛋白激酶家族。谷子SiMAPK3三级结构与拟南芥MAPK具有很高的相似度，存在着11个丝氨酸、8个苏氨酸、4个酪氨酸及大量潜在磷酸化位点。RT-qPCR分析表明，SiMAPK3在拔节前期谷子根、茎和叶中均有表达，其在叶片中的表达量最高，约为其在根中表达量的25倍。SiMAPK3响应了低温（4℃）、高盐（300 mmol/L NaCl）、干旱和ABA（200 μmol/L）、JA（200 μmol/L）的胁迫。在低温胁迫12 h后，SiMAPK3的表达量上调了16倍，在高盐胁迫3 h后，SiMAPK3的表达量上调了8倍。谷子SiMAPK3基因的克隆及功能分析，为进一步解析谷子叶片生长发育及响应低温和高盐等胁迫信号转导过程提供重要依据。

关键词: 谷子, SiMAPK3, 叶片发育, 低温胁迫, 高盐胁迫, 脱落酸, 茉莉酸甲酯, 干旱胁迫

Abstract:

Mitogen-activated protein kinases（MAPKs）are a class of serine/threonine protein kinases that play important roles in plant growth and development, response to stress, and hormone signal transduction. In this study, the SiMAPK3 gene, which was the highest protein homology to AtMAPK3 in Arabidopsis, was cloned from the foxtail millet variety Yugu 1. The physicochemical properties, structure and function of SiMAPK3 protein were systematically analyzed by bioinformatics methods. The expressions of SiMAPK3 gene in different tissues and different abiotic stress in the early shooting stage of the foxtail millet was analyzed by RT-qPCR. The results showed that the open reading frame of SiMAPK3 gene was 1 128 bp, encoding a deduced protein of 376 amino acids, with a predicted molecular weight of 43 427.85 Da and an isoelectric point of 5.46. The SiMAPK3 in the foxtail millet was a hydrophilic extramembrane protein without signal peptide. Its secondary structure included 44.27% α helix, 14.67% β sheet, 5.07% extended chain and 36.00% random coil. SiMAPK3 amino acid 44-328 contained a Pkinase conserved domain, belonging to the MAPK protein kinase family. The tertiary structure of SiMAPK3 had high similarity with Arabidopsis MAPK protein, SiMAPK3 contained 11 serines, 8 threonines, 4 tyrosines and a large number of potential phosphorylation sites. Protein structural analysis showed that it had a conserved PKinase domain. RT-qPCR analysis showed that SiMAPK3 was expressed in the roots, stems and leaves of foxtail millet in the early shooting stage, and its expression level was the highest in leaves, which was about 25 times that in roots. SiMAPK3 responded to low temperature（4℃）, high salt（300 mmol/L NaCl）, drought and ABA（200 μmol/L）and JA（200 μmol/L）and other stresses. After 12 h of low temperature, the expression of SiMAPK3 was up-regulated 16-fold, and after 3 h of high-salt, the expression of SiMAPK3 was up-regulated 8-fold. The cloning and functional analysis of the SiMAPK3 gene provide an important basis for further analysis of the leaves development and the signal transduction in response to low temperature and high salt in foxtail millet.

Key words: Setaria italica, SiMAPK3, leaf development, cold stress, salt stress, ABA, JA, drought stress

王海龙, 李雨倩, 王勃, 邢国芳, 张杰伟. 谷子SiMAPK3基因的克隆和表达特性分析[J]. 生物技术通报, 2023, 39(3): 123-132.

WANG Hai-long, LI Yu-qian, WANG Bo, XING Guo-fang, ZHANG Jie-wei. Isolation and Expression Analysis of SiMAPK3 in Setaria italica L.[J]. Biotechnology Bulletin, 2023, 39(3): 123-132.

图/表 5

图1 谷子SiMAPK3的序列与结构分析 A：谷子SiMAPK3蛋白的疏水性；B：谷子SiMAPK3二级结构分析；C：谷子SiMAPK3结构域分析；D：谷子SiMAPK3三级结构模型；E：谷子SiMAPK3磷酸化位点预测

Fig. 1 Sequence and structure analysis of SiMAPK3 in foxtail millet A：Hydrophobicity of SiMAPK3. B：Analysis of the SiMAPK3 secondary structure. C：Analysis of the SiMAPK3 domain. D：The tertiary structure model of SiMAPK3. E：Prediction of phosphorylation sites in SiMAPK3

图2 SiMAPK3与其他物种MAPK蛋白的多重序列比对红框表示高度保守的TEY特征序列

Fig. 2 Multiple sequence alignment of the SiMAPK3 and MAPK proteins from other species Red box indicates the specific sequence of highly conservative TEY

图3 SiMAPK3与其他物种MAPK蛋白的系统进化树分析

Fig. 3 Phylogenetic tree analysis of the SiMAPK3 and MAPK proteins from other species

图4 谷子SiMAPK3基因在拔节前期各组织中的表达分析 RT-qPCR检测SiMAPK3基因的表达水平, 以谷子SiActin作为内参基因。n=3，**：极显著性差异（P<0.01）。下同

Fig. 4 Expression analysis of SiMAPK3 in different tissues of S. italica at the shooting stage of foxtail millet RT-qPCR analysis of SiMAPK3 gene expression in various organs, the SiActin in S. italica was used as an internal reference. n=3. Double asterisks（**）in each column indicate a significant difference at P<0.01 level. The same below

图5 SiMAPK3基因在不同处理下的表达模式 A：干旱胁迫下SiMAPK3基因的表达；B：低温胁迫下SiMAPK3基因的表达；C：20% PEG6000胁迫下SiMAPK3基因的表达；D：300 μmol/L高盐胁迫下SiMAPK3基因的表达；E：200 mmol/L脱落酸胁迫下SiMAPK3基因的表达；F：200 μmol/L 茉莉酸胁迫下SiMAPK3基因的表达。*：差异性显著（P<0.05）

Fig. 5 SiMAPK3 expression patterns under different treatments A: SiMAPK3 gene expression under dehydration; B: SiMAPK3 gene expression under 4℃; C: SiMAPK3 gene expression under 20% PEG6000; D: SiMAPK3 gene expression under 300 mmol/L NaCl; E: SiMAPK3 gene expression under 200 μmol/L ABA; F: SiMAPK3 gene expression under 200 μmol/L JA. Asterisk（*）indicates a difference at P<0.05 level

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