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为了探究托品酮还原酶(tropinone reductase,TR)基因在石斛中的功能,该研究采用RT-PCR结合RACE技术,以霍山石斛原球茎为材料,克隆得到霍山石斛TR-I基因的cDNA序列,其在Gen Bank中的登录号为KY912085。TR-I基因的cDNA长1 043 bp,包含一个完整的共807 bp的ORF(open reading frame)(79-885),共编码268个氨基酸。生物信息学分析表明,该蛋白质可能是一种疏水性稳定蛋白质,无信号肽,不含卷曲螺旋结构,具有14个功能位点、47个潜在磷酸化位点,其二级结构由螺旋、折叠和卷曲结构组成。系统进化树分析表明,霍山石斛TR-I与铁皮石斛、金钗石斛的亲缘关系较近,处于同一分支。qPCR结果显示,TR-I基因在霍山石斛不同生长期的相对表达量均表现为茎>叶>根,且茎和叶中TR-I的表达量均随着生长期的延长呈现低–高–低的变化趋势;在不同品种中,金钗石斛的TR-I基因相对表达量最高,铁皮石斛中最低;不同浓度茉莉酸甲酯(methyl jasmonate,Me JA)处理比较,100μmol/L茉莉酸甲酯处理下TR-I基因表达量最高,推测该基因可能参与霍山石斛生物碱的合成途径。
In order to explore the function of tropinone reductase (TR) gene in Dendrobium, the cDNA sequence of TR-I gene from Dendrobium huoshanense was cloned by RT-PCR and RACE technique using the protocorm-like of Dendrobium huoshanense as material. Its accession number in Gen Bank is KY912085. The cDNA of TR-I gene is 1 043 bp in length and contains a complete ORF (open reading frame) of 807 bp (79-885) encoding a total of 268 amino acids. Bioinformatics analysis showed that this protein may be a hydrophobic stable protein, no signal peptide, no coiled-coil structure, 14 functional sites and 47 potential phosphorylation sites. The secondary structure consists of helix, And curly structure. Phylogenetic tree analysis showed that Dendrobium huoshanense TR-I was closely related to Dendrobium candidum and Dendrobium nobile, and was on the same branch. The results of qPCR showed that the relative expression of TR-I gene in different stages of D. huoshanense showed stem> leaf> root, and the expression of TR-I in stem and leaf showed low-high- The highest relative expression level of TR-I gene was found in Dendrobium nobile, and the lowest was in Dendrobium candidum. Under different concentrations of methyl jasmonate (Me JA) treatment, 100μmol / L jasmonate A Under the ester treatment, the expression of TR-I gene was the highest, suggesting that the gene may be involved in the synthesis of Dendrobium huoshanense alkaloids.