Isolation And Characterization Of Caffeic Acid O-methyl-transferase Gene From Larix Gmelinii

Larix gmelinii, a high deciduous tree, is widely distributed in Greater Hinggan moutains region of China to serve as the dominant tree species for afforestation there. With strong resistance, Larix gmelini’s wood material is good, hence its extensive application value. Caffeic acid-3-O-methl transferase (COMT) is an important methylase in plant lignin biosynthesis pathway, which plays an important role in biosynthesis of syringyl lignin monomer. To control expression of this gene can influence content of lignin and composition of monomer. Therefore, to research characteristics and control mechanism of LgCOMT gene can provide reference for improvement on materials quality of forest.In this study, Full-length sequence of Larix gmelinii Caffeic acid O-methyl transferase gene (LgCOMT) was cloned using degenerate-polymerase chain reaction (PCR) and rapid ampliication of cDNA ends (RACE) techniques. The cloned LgCOMT cDNA contains 1173 bp, which has an open reading frame (ORF) of 1092 bp encoding a protein of 364 amino acids,77 bp of 5’-untranslated region (UTR) and 47 bp of 3’-UTR, and protein’s molecular weight is 40.18kDa. The theoretical isoelectric point is 5.63. Full length of genomic DNA of the LgCOMT contains 1400 bp, composed of three extrons (respectively 759bp,65bp and 349bp) and two introns (respectively 91bp and 136bp). The bioinformatics analysis showed LgCOMT belongs to methyltransferase (OMTs) family, and the sequence contains a conserved element specific to OMTs family. The analysis of system evolution showed a close genetic relation with gymnosperm COMTs, such as Larix kaempferi, Picea abies, and gingko biloba, etc.Semi-quantitative RT-PCR analytic result showed that LgCOMT is mainly expressed in stems and cone of L. gmelinii seedlings, with less expression in root. LgCOMT gene is transformed to prokaryote colibacillus, and subjected to IPTG inducing expression to get fusion protein expression with size conforming to anticipation. Besides, pBI101-LgCOMT, the plant expression vector for gene is successfully constructed, which lays a foundation for analyzing the gene’s functional characteristics and utilizing the gene to research plant genetic transformation.The above findings provides theoretical and experiment reference for further researching the regulation mechanism of LgCOMT in lignin monomer biosynthesis and improvement on forest wood using the gene.