Full-length CDNA And Genomic DNA Cloning Of Caffeoyl Coenzyme A-O-methyltransferase From Caragana Korshinkii Kom And Its Bioinformatics Analysis

Lignin is a major structural component of plant secondary cell walls and is one of the most abundant organic polymer on earth. The lignin has important influence on plant itself and humanity's life and production.Caffeoyl coenzyme A O-methyltransferase (CCoAOMT) is one kind of methyltransferase which plays an important role in lignin biosynthesis.In this research, we had cloned two full-length cDNA sequences which were named CkCCoAOMT-C and CkCCoAOMT-G and one full-length genomic DNA sequence.The two full-length cDNA sequences were cloned by RT-PCR and RACE technology from Caragana Korshinskii Kom. We speculated that they belonged to the CCoAOMT gene family of Caragana Korshinskii Kom. The sequence of the genomic DNA contains five exons and four introns.Bioinformatics assay showed that CCoAOMT-C and CCoAOMT-G are hydrophilic protens; their molecular weight are about 28,012.12D and 27,970.08D respectively and isoelectric points are aound 5.55 and 5.54 respectively; both protens are composed of 247 amino acid residues. The 125th and 134th amino acid residues of CCoAOMT-C are different from those of CkCCoAOMT-G; the secondary structure and tertiary structure of CCoAOMT-C are also different from CkCCoAOMT-G; both CCoAOMT-C and CCoAOMT-G have Methyltransf₃ domain and SAM binding site, so they are thought to belong to the family of O-methyltransferases.We constructed the overexpression binary vector pCHF3 containing CkCCoAOMT-C and CkCCoAOMT-G respectively. Totally, three homozygous CkCCoAOMT-C overexpression transgenic lines and six homozygous CkCCoAOMT-G overexpression transgenic lines were obtained by screening on 1/2MS media containing kanamycin. And the target gene expression level in all the homozygous lines was detected by semi-quantitative RT-PCR and proved to be overexpressed at different level.