当归DXR基因保守区克隆和组织特异性表达分析  

Cloning and tissue-specific expression analysis of conserved fragments of DXR gene from Angelica sinensis

作　　者：雒军[1,2] 王引权[1,2] 温随超 张金林[3] 夏琦[1] 

机构地区：[1]甘肃中医学院,甘肃兰州730000 [2]甘肃省高校中(藏)药化学与质量研究省级重点实验室,甘肃兰州730000 [3]兰州大学草地农业科技学院,甘肃兰州730020

出　　处：《中草药》2014年第13期1907-1913,共7页Chinese Traditional and Herbal Drugs

基　　金：国家自然科学基金资助项目(81060327;81260616)

摘　　要：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 conserved cDNA fragment of 1-deoxy-D-xylulose 5-phosphate reductoisomerase（DXR） gene from Angelica sinensis and to analyze its tissue-specific expression in roots,stems,and leaves.Methods A pair of degenerate primers were designed according to the conservative sequences of the cloned DXR from other plant species.The total RNA from the leaves of A.sinensis was as template,DXR fragment was obtained by reverse transcription polymerase chain reaction（RT-PCR）,connected to pGEM-T Easy vector then transformed into Escherichia coli DH5α.The positive clone identified by PCR was sequenced.Taking Actin of A.sinensis as a reference gene,SYBR Green quantitative RT-PCR was used to detect the relative expression levels of DXR different tissues of A.sinensis.Results The DXR fragment of A.sinensis containing 564 bp encoding 187 amino acids was registered in Genbank（No.KJ000259）.The sequence identity analysis suggested that both the obtained nucleotide sequence and its corresponding amino acid sequence shared more than 80% of homology with GenBank DXRs from Gossypium barbadense and other five higher plant species.DXR was expressed at the highest level in the leaves,and the relative expression levels in the leaves were 2.5 and 3.2 times relative to the stems and roots,respectively（P 0.01）.Conclusion A novel DXR fragment is cloned from A.sinensis and its tissue-specific expression in A.sinensis is investigated.This work might establish an experimental basis for the effective application of DXR in the future.

关 键 词：当归 1-脱氧-D-木酮糖-5-磷酸还原异构酶 克隆 肌动蛋白 RT-PCR 

分 类 号：R282.12[医药卫生—中药学]