Cloning and expression analysis of phosphomevalonate kinase gene PcoPMK in Prunus conradinae
[Objective]The objective of this study was to clone the cDNA sequence of the phosphomevalonate kinase gene of Prunus conradinae,and analyze the expression level of this gene in the petals of the'YS01'at different developmental stages,as well as the structural characteristics and physicochemical properties of the encoded protein,so as to provide a reference for further research on the function of PcoPMK gene in the synthesis pathway of terpenoids in P.conradinae.[Method]The specific primers had been designed with reference to the conserved cDNA sequence of homologous PMK gene of related species.The cDNA sequence of the PcoPMK gene was amplified using RT-PCR technology,and cloned for sequencing.In addition,quantitative RT-PCR technology was used to analyze the expression level of the gene in the petals of'YS01'at different developmental stages.ORF Finder online tool was used to predict gene open reading frame and encoded protein sequence.The physicochemical properties,secondary structures,tertiary structures,and functional domains of the PcoPMK protein were analyzed using ExPASy,SOPMA,Swiss model,and CDD online tools,respectively.The transmembrane structure,signal peptide and subcellular localization of the PcoPMK protein were predicted by DeepTMHMM,SINALP 4.0 Server and PredictProtein online programs,respectively.The phylogenetic tree of the PcoPMK gene was constructed using MEGA7.0 software.[Result]The open reading frame sequence of PcoPMK gene in P.conradinae was 1 530 bp in length,encoding 509 amino acids.PcoPMK protein belonged to hydrophilic stable protein without transmembrane helix and signal peptide,which was located in the cytoplasm.Its relative molecular weight was 55.199 kD,and theoretical isoelectric point was 5.68.The PcoPMK protein had high sequence similarity with PMK homologous proteins of representative species,such as almond,tobacco,and arabidopsis.It contained three typical and conservative domains,and one ATP binding site.The relative expression level of the PcoPMK gene in the petals of the full opening stage was significantly higher than that in bud and half opening stage in P.conradinae'YS01'(P＜0.05),which was consistent with the trend of changes in the content of aromatic terpenes in the petals.[Conclusion]In this study,the PcoPMK gene is successfully cloned,and it is speculated that it may play an important role in the synthesis pathway of terpenes in the petals of P.conradinae'YS01'.These results will provide a reference for further research on the biological function of PcoPMK gene in P.conradinae.
万方数据
维普
