Cloning Of Ramie COMT And CCoAOMT Gene And Construction Of Their Antisence Expression Vectors

Lignin is, second to cellulose, the most abundant organic compound in the terrestrial biosphere. The importance of lignin ranges from its fundamental roles in the evolution of land plants, global carbon cycling, and development, to its role in the biotic and abiotic stress resistance of plants. However, its presence in plants has negative effects on pulp and paper industry, ramie textile industry and stockbreeding. Therefore, it has been the focus to study lignin biosynthesis and genetic manipulation.Boehmeria nivea L. is a kind of excellent textile material. What we utilize is its phloem fiber. The raw fiber must be unglued before it is used to spin. During the process of ungluing, the lignin is the impurity which is most difficult to be extracted. It increases not only the cost, but also the pollution. To regulate the expression of the genes of key enzymes by molecular and genetic approaches will result in the reduction of lignin contents or modification of the lignin composition. The development of such new ramie resources can cut down the costs and reduce the pollution from the origin.The results of this paper include the molecular cloning of the caffeic acid/ 5-hydroxy-ferulic acid O-methytransferase (COMT) gene and cafffeoyl-CoA 3-O-methytransferase (CCoAOMT) gene, and the construction of the phloem-specific antisence expression vector.A method based on degenerate PCR and rapid amplification of cDNA ends (RACE) for cloning a full-length cDNA is described. This method is more simple and inexpensive than screening cDNA library, and can be easily adapted to clone other genes. In this paper, the full-length cDNA of COMT gene of Boehmeria nivea L. was cloned by this method. The size of the cDNA fragment is 1322bp, which included a 1098bp open reading frame and encoded a peptide of 365 amino acids. The result of BLAST in NCBI shows that its sequence of cDNA exhibits 82% homology with Populus amygdalus. The deduced amino acid sequence exhibits 93% homology with Prunus dulcis,contain the conserved domain of O-methyltransferase-â…¡superfamily. The seven characteristic motifs of plant OMT-â…¡superfamily are well conserved in the ramie COMT.