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目的克隆当归Angelica sinensis 1-脱氧-D-木酮糖-5-磷酸还原异构酶(DXR)编码基因部分保守区序列,分析DXR基因在当归根、茎、叶中的特异性表达。方法根据已克隆的植物DXR保守序列设计一对简并引物,以当归叶片总RNA为模板,采用RT-PCR方法扩增出DXR片段并连接到pGEM-T Easy载体上,转化大肠杆菌菌株Escherichia coli DH5α,阳性克隆经PCR检测后测序。运用RT-qPCR方法,以当归肌动蛋白基因Actin为内参基因,检测DXR在当归不同组织中的表达量。结果得到一段564 bp的DXR基因序列,并在GenBank注册(登录号:KJ000259)。序列分析表明,该片段编码187个氨基酸,与GenBank中注册的海岛棉Gossypium barbadense等6个物种的DXR核苷酸序列同源性和氨基酸序列同源性均在80%以上;DXR在叶中表达量最高,相对表达量分别是茎和根组织中的2.5倍和3.2倍(P<0.01)。结论本研究首次从当归中克隆出了DXR部分保守区序列,并揭示了DXR在不同组织中的差异表达,为有效利用该基因奠定前期工作基础。
OBJECTIVE: To clone the partial conserved region of the gene encoding Angelica sinensis 1-deoxy-D-xylulose-5-phosphate reductoisomerase (DXR) and analyze its specific expression in root, stem and leaf of Angelica sinensis. Methods A pair of degenerate primers was designed according to the conserved sequence of the cloned plant DXR. The total RNA of Angelica keiskei was used as template. The DXR fragment was amplified by RT-PCR and ligated into pGEM-T Easy vector and transformed into E. coli strain Escherichia coli DH5α, positive clones were sequenced after PCR detection. RT-qPCR method was used to detect the expression of DXR in different tissues of Angelica Sinensis (Angelica sinensis) by using actin gene Actin as an internal control gene. The results obtained a 564 bp DXR gene sequence, and registered in GenBank (accession number: KJ000259). Sequence analysis showed that the fragment encoded 187 amino acids and shared 80% homology with the DXR nucleotide sequences of 6 species of Gossypium barbadense registered in GenBank. DXR was expressed in leaves The highest amount and relative expression were 2.5 and 3.2 times higher in stem and root tissues (P <0.01). Conclusions This study, for the first time, cloned part of the conserved region of DXR from Angelica sinensis and revealed the differential expression of DXR in different tissues, which laid the foundation for the effective utilization of this gene.