以苎麻[Boehmeria nivea（L.）Gaud]为材料，研究木质素代谢的关键酶苎麻咖啡酰辅酶A甲基转移酶基因序列。分离了高纯度RNA和DNA。以RNA为模板，用简并引物以RT-PCR的方法，克隆了苎麻CCoAOMT基因cDNA部分序列，长度为486 bp，编码162个氨基酸残基。此cDNA序列为苎麻植物中首次克隆的(caffeoyl-CoA 3-O-methyltransferase，CCoAOMT)新基因序列。GenBank注册，登录号为AY651026。以苎麻4个栽培品种基因组DNA为模板，经温度梯度PCR条件优化，获得苎麻CCoAOMT基因的部分基因组序列，GenBank注册号为AY818191 (湘苎3号，922 bp)、AY822619 (圆麻，915 bp)、AY822620 (芦竹青，914 bp)、AY822622 (青麻，914 bp)。生物信息学分析结果发现，苎麻4个栽培品种的CCoAOMT基因基因组DNA片段序列有一定的差异。
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 24th 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.
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