Cloning and expression analysis of O-methyltransferase genes in Musella lasiocarpa
Objective To clone the O-methyltransferase(OMT)genes in Musella lasiocarpa and analyze their bioinformatics information and expression patterns.Methods Three MlOMTs enzyme genes(MlOMT1,MlOMT2 and MlOMT3)were screened based on the transcriptomic data of M.lasiocarpa and prokaryotically expressed.Online software were used for bioinformatics analysis.Phylogenetic tree was constructed using MEGA11.Real-time fluorescence quantitative PCR was conducted to detect gene expression patterns.Results Three MlOMTs were cloned by PCR.The lengths of the three M1OMT proteins ranged from 289 to 400 amino acids,and the range of the predicted molecular weight was from 3 147 360 to 4 526 756.Three MlOMTs were unstable hydrophilic proteins.Both MlOMT1 and MlOMT2 lacked transmembrane regions,whereas MlOMT3 had two transmembrane helices with its N-terminal located in the cytoplasm.All the three proteins were mainly localized in chloroplasts,without signaling peptides.They mainly consisted of α-helical and irregular coiled structures,and contained conserved domains of the SAM-dependent methyltransferase superfamily.Phylogenetic analysis revealed that M1OMT1 clustered with Hv7OMT from Hordeum vulgare and OsOMT17 from Oryza sativa subsp.Japonica,while MlOMT2 clustered with McPFOMT from Mesembryanthemum crystallinum.Additionally,MlOMT3 clustered together with AtCCoAOMT1 from Arabidopsis thaliana.The close relationships mentioned above suggested they might have similar biological functions.The soluble protein expression of recombinant vectors pMal-c4X-M1OMT1 and pMal-c4X-MlOMT2 in BL21(DE3)was achieved through preliminary optimization of the expression conditions.Tissue-specific expression analysis showed that relative expression levels of MlOMT1 varied across different tissues as follows:leaves＞bracts＞seeds;whereas relative expression levels of MlOMT2 and MlOMT3 were found as bracts＞seeds＞leaves.Conclusion The cloning,bioinformatics analysis and expression analysis of three MlOMTs of M.lasiocarpa was completed,which lays the foundation for characterizing their functions.
Musella lasiocarpa(Franch.)C.Y.Wu ex H.W.Limethyltransferasegene cloningquantitative real-time PCRbioinformatics analysis
万方数据
维普
