关键词: 苎麻[Boehmeria nivea（L.）Gaud], CCoAOMT基因, 基因克隆

Abstract:

The objective of this study was to examine the sequence of ramie CCoAOMT and investigate ramie lignin biosynthesis pathway. High quality RNA(Fig.1) and DNA were extracted from ramie [Boehmeria nivea（L.）Gaud] and used as a template for cloning the caffeoyl-CoA 3-O-methyltransferase gene cDNA by RT-PCR with degenerate primers under gradient temperature. Colonies with target foreign DNA inserts was identified by PCR (Fig.2). Recombinant pMD18-T plasmid was identified by restriction digestion (Fig.3) and PCR (Fig.4), also sequence analysis. Ramie caffeoyl-CoA 3-O-methyltransferase gene cDNA partial sequence with GenBank accession number AY651026 is 486 bp long, coding 162 amino acids. The result of the domain analysis showed the ramie CCoAOMT to be a member of the O-methyltransferases family. CCoAOMT gene was first discovered in Boehmeria nivea（L.）Gaud and was first documented and recorded as a part of the Urticaceae family.
The new partial genomic sequences of the cloned ramie CCoAOMT gene resulted in the new GenBank accession numbers: AY818191 (Xiangzhu 3, 922 bp), AY822619 (Yuanma, 915 bp), AY822620 (Luzhuqing, 914 bp) and AY822622 (Qingma, 914 bp). The results of bioinformatic analysis showed that the four sequences were different in length and had three introns each. In each of the four sequences the first introns are different in length, the second in some base mutations, and the third in both length and base mutation.
Future research is necessary in order to study and see if these differences cause the differences in the gene expression or not. There were only 5 different bases in the extron of the 4 sequences. Corresponding to the cDNA sequence, in genomic sequences, in the 24^th base, the base mutation did not cause an amino acid change, Pro was coden; in the 185 ^th base, the base mutation caused the changes of amino acid, resulting in Thr and Asn coden with two distinct quality; in the 188 ^th base, the base mutation caused the changes of amino acid resulting in Ala and Gly coden, which was similar in quality; in the 337 ^th base, the base mutation caused the changs of amino acid resulting in Ile and Leu coden which was similar in molecular consistency but different in structure; lastly, in the 423 ^th base, the base mutation did not cause the changes of amino acid coden and remained Leu coden. Future research is also necesary in order to determine if these amino acid changes caused enzyme activity changes or not.

Key words: Ramie [Boehmeria nivea（L.）Gaud], CCoAOMT, Gene cloning