Molecular Cloning of Longan Caffeoyl-CoA O-methyltransferase (DLCCoAOMT) and Its Expression Analysis

【Objective】The aim of this study was to clone full-length cDNA of a key enzyme gene DLCCoAOMT related to lignin metabolism in longan,investigate its sequence characteristics and analyze its expression in different organs under low temperature stress,as well as expression in the prokaryote.【Method】 The DLCCoAOMT gene cDNA sequence was cloned from longan leaf using RT-PCR and RACE techniques.The bioinformatics methods were used to analyze putative amino acid sequence and real-time PCR method were used to study the expression of DLCCoAOMT gene in different tissues.【Result】 The full-length cDNA of DLCCoAOMT(GenBank accession number: JN093023) in longan was cloned.The sequence consists of 993 bp with an open reading frame of 744 bp,encoding a polypeptide of 247 amino acids.Homology analysis showed that the deduced DLCCoAOMT protein was highly homologous to other CCoAOMT proteins from different species.Phylogenetic analysis also indicated that DLCCoAOMT was very closely related to DLCCoAOMT of Betula.Real-time PCR results showed that the DLCCoAOMT expressed in root,stem and leaf,and its expression was different among three organs.The mRNA of DLCCoAOMT was abundant in root and stem,but less in leaf.Furthermore,DLCCoAOMT transcription level was significantly different among root,stem and leaf with time courses of chilling treatment.Prokaryotic expression showed that recombinant plasmid was efficiently expressed in Escherichia coli BL21.【Conclusion】 Gene DLCCoAOMT was firstly isolated and characterized from longan,which may be involved in chilling stress.