Cloning and expression analysis of CsMAPKK3 gene in tea plant

【Objective】The CsMAPKK3 gene of tea tree was cloned and its expression was analyzed to provide theoretical support for clarifying the biological function of tea plant CsMAPKK3 gene and revealing its role in tea plant's stress resistance.【Method】In this study, the full-length cDNA of MAPKK3 gene were cloned using RT-PCR from Tieguanyin tea plant. Then, the bioinformatics characteristics and functions of CsMAPKK3 gene were predicted, and its expression patterns in various tissues and under different stress treatments were investigated using real-time fluorescence quantitative PCR.【Result】The full-length cDNA of CsMAPKK3 was 1941 bp, with a 1557 bp open reading frame(ORF), encoding 518 amino acids, and submitted to the GenBank with accession number of AUD40506.1. The molecular weight of CsMAPKK3 protein was 57.52 kD, and the theoretical isoelectric point(pI) was 5.39. The amino acid sequence prediction showed that it was an unstable protein without transmembrane helix structure and signal peptide, and contained multiple phosphorylation sites. CsMAPKK3 protein, located in cytoplasm and nucleus, belonged to PKC type MAPKK, and had catalytic domains such as SPS1, S_TKC, D(I/L/V) K activation domain and S/T-X_3-5-S/T conserved domain. It had close relationship with Chinese kiwifruit(PSS35243.1), and was clustered in a class with Arabidopsis B subfamily in phylogenetic tree. Additionally, promoter sequence analysis showed that the 1714 bp promoter region in the initiation codon upstream of CsMAPKK3 contained several cis-acting elements related to light response, stress response, plant hormones, and anaerobic induction. Real time quantitative PCR analysis showed that CsMAPKK3 was expressed in roots, stems, leaves, flowers and fruits, and the expression level was higher in stems and fruits, which was significantly higher than that in flowers(P<0.05), but had no significant difference with that in roots and leaves(P>0.05). The expression of CsMAPKK3 was down-regulated by cold stress, but up-regulated by abscisic acid(ABA), salt and drought treatments.【Conclusion】The expression of CsMAPKK3 is tissue-specific, and it is involved in the tea plant response to ABA, high salt, low temperature and drought stresses.