Abstract: 【Objective】The objective of this study is to clone serine/threonine protein kinase VvCIPK10 in grapevine, and analyze the kinase characteristics and its expression pattern under stresses. This study will provide a theoretical basis for further study on the molecular function of VvCIPK10 involved in abiotic stress, and investigate the molecular mechanism of grapevine stress resistance. 【Method】The sequences of VvCIPK10 were obtained by electronic cloning technology, and the specific primers were designed to perform RT-PCR reaction. Open reading frame and conserved structure domain of VvCIPK10 were analyzed. The prokaryotic expression vector was constructed and transformed into the expression cells, and the recombinant bacteria were induced by IPTG. The expression products were collected and the protein sample was prepared. The protein samples were analyzed by SDS-PAGE electrophoresis, and the soluble characteristics of the fusion protein were analyzed. The fusion protein was induced by IPTG. The cells were collected and broken by using ultrasonic. The MBP-VvCIPK10 fusion protein was purified by maltose binding protein purification column and analyzed by SDS-PAGE. The purified fusion protein was incubated with in vitro self-phosphorylation buffer solution. After SDS-PAGE electrophoresis, the phosphor screen was performed and the phosphorylation reaction was detected in vitro. The recombinant transient expression vector pBI221-GFP/VvCIPK10 was constructed. The recombinant expression vector pBI221-GFP/VvCIPK10 was transformed into protoplasts by PEG mediated transient transformation. The recombinant expression vector pBI221-GFP/VvCIPK10 was transformed into onion epidermal cells by gene gun mediated transformation, and the fluorescence signal was detected by laser scanning confocal microscope after 16 h of culture. The relatively consistent and robust grape plants were selected, samples were taken at different times after drought, low temperature and salt stress treatments, at the same time, different tissue samples of grapes were taken in the field, with a kit to extract total RNA. After reverse transcription, expression of VvCIPK10 was detected by real-time quantitative PCR. 【Result】The full-length of VvCIPK10 is 1 357 bp, 5′ end of non-coding region is 30 bp, 3′ end of non-coding region is 156 bp, the open reading frame is 1 171 bp. VvCIPK10 open reading frame encoded 436 amino acids, the theoretical isoelectric point is 8.59, molecular weight is 48.7 kDa. Conserved domain prediction analysis showed that the protein has a kinase domain in 5′ terminal, a PPI domain and a NAF domain in 3′ terminal. BLSATP analysis showed VvCIPK10 consistency with peach CIPK (XP_007205151) is highest (74%). Recombinant expression vector pMAL-C5X/VvCIPK10 transformation in Escherichia coli, expressed the molecular weight of fusion protein is consistent with the predicted molecular weight (43 kDa+48.7 kDa). MBP-VvCIPK10 fusion protein was purified by column. VvCIPK10 autophosphorylation activity was dependent on Mn²⁺ but not dependent on Mg²⁺ and Ca²⁺, and EDTA could inhibit the autophosphorylation activity of the VvCIPK10. Subcellular localization showed VvCIPK10 in the nucleus, cell membrane and cytoplasm. VvCIPK10 expressed in various tissues of grapevine. VvCIPK10 transcripts mainly accumulated in grapevine roots and leaves, but showed low expression levels in grapevine stem, inflorescence, fruit and tendril. After drought, low temperature and salt stress treatments, VvCIPK10 showed the induced expression model. The expression of VvCIPK10 reached peak value at 6 h after low temperature stress, and reached the peak at 2 h after drought and salt stresses. 【Conclusion】It was concluded that grapevine VvCIPK10 as a serine threonine protein kinase is able to respond to drought, low temperature and salt stress, suggesting that VvCIPK10 plays an important role in resistance to abiotic stress.

Key words: grapevine, calmodulin like B subunit, serine threonine protein kinase, VvCIPK10, expression analysis