水曲柳结构域重新甲基化酶DRM基因片段的克隆及同源性分析

Molecular Cloning and Sequence Analysis of DRM Gene Segment of Fraxinus mandschurica

[目的]克隆并分析水曲柳结构域重新甲基化酶(DRM)基因片段。[方法]根据GenBank上已发表的DRM氨基酸序列,采取CODEHOP方法设计合成1对简并引物,采用RT-PCR技术对水曲柳DRM基因进行扩增,将PCR产物与pMD 18-T连接后转化至E.coli JM109感受态细胞,检测阳性克隆,测序并进行序列分析。[结果]克隆得到的DRM基因cDNA序列长802 bp,由267个氨基酸残基组成,命名为FmadrmA。序列比对表明FmadrmA与参考的12个物种的DRM在氨基酸水平上表现出较高的同源性,其与山葡萄、番茄、黄瓜、野草莓、大豆、苜蓿、玉米、烟草、拟南芥、毛果杨、水稻和乌拉尔图小麦的DRM同源性分别为61%、54%、51%、50%、60%、48%、43%、44%、37%、42%、42%和39%。系统进化树分析表明同源的DRM蛋白可大致聚为3类,而水曲柳与其他植物的亲缘关系较远。[结论]该试验成功获得了水曲柳FmadrmA基因片段,并对其进行了同源性分析,为进一步研究结构域重新甲基化酶基因及分析其表达特性奠定了基础。

[ Objective ] The aim was to clone and analyze the domain remethylation enzyme （DRM） gene fragment of Fraxinus mandshurica. [ Method] According to the sequence of DRM amino acid published in GenBank, a pair of degenerate primers was designed and synthesized by the CODEHOP method. DRM gene of F. mandshurica was amplified by RT-PCR technique. The PCR product was connected with pMD18 - T and transformed into competent cells of E. coli JM109. Then the positive clone was identified and the sequence was sequenced and analyze. [ Result] The cDNA sequences of cloned DRM gene with named FmadrmA was 802 bp in length, and coded 267 amino acid residues. Sequence alignment showed that FmadrmA presented high homology with other 12 species＇ DRM at the level of amino acid. Compared with the corresponding region of grape, tomato, cucumber, wild strawberry, soybean, alfalfa, corn,tobacco, Arabidopsis, poplar, rice and wheat, the protein homology of F. mand- shurica was 61% ,54% ,51% ,50% ,60% ,48% ,43% ,44% ,37% ,42% ,42% and 39% ,respectively. Phylogenetic tree analysis indicated that the DRM proteins with homology could be roughly classified into three categories, and F. mandshurica had a far genetic relation with other palntts. [ Conclusion ] The FmadrmA gene fragment of F. mandshurica was obtained successfully, and the homology was analyzed. It laid the foundation for the further research on domain remethylase gene and analysis of its expression characteristics.