Isolation and Expression Analysis of SiMAPK3 in Setaria italica L.

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

Abstract

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

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.

Figures/Tables 5

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

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

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

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

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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