郑汉, 荆礼, 姚娜, 杨青山, 彭华胜, 申业, 黄璐琦. 香樟1-脱氧-D-木酮糖-5-磷酸还原异构酶基因CcDXR1的克隆和表达分析J. 药学学报, 2016,51(9): 1494-1501. doi: 10.16438/j.0513-4870.2016-0225
引用本文: 郑汉, 荆礼, 姚娜, 杨青山, 彭华胜, 申业, 黄璐琦. 香樟1-脱氧-D-木酮糖-5-磷酸还原异构酶基因CcDXR1的克隆和表达分析J. 药学学报, 2016,51(9): 1494-1501. doi: 10.16438/j.0513-4870.2016-0225
ZHENG Han, JING Li, YAO Na, YANG Qing-shan, PENG Hua-sheng, SHEN Ye, HUANG Lu-qi. Cloning and expression analysis of 1-deoxy-D-xylulose-5-phosphate reductoisomerase gene (CcDXR1)in Cinnamomum camphora (L.) PreslJ. Acta Pharmaceutica Sinica, 2016,51(9): 1494-1501. doi: 10.16438/j.0513-4870.2016-0225
Citation: ZHENG Han, JING Li, YAO Na, YANG Qing-shan, PENG Hua-sheng, SHEN Ye, HUANG Lu-qi. Cloning and expression analysis of 1-deoxy-D-xylulose-5-phosphate reductoisomerase gene (CcDXR1)in Cinnamomum camphora (L.) PreslJ. Acta Pharmaceutica Sinica, 2016,51(9): 1494-1501. doi: 10.16438/j.0513-4870.2016-0225

香樟1-脱氧-D-木酮糖-5-磷酸还原异构酶基因CcDXR1的克隆和表达分析

Cloning and expression analysis of 1-deoxy-D-xylulose-5-phosphate reductoisomerase gene (CcDXR1)in Cinnamomum camphora (L.) Presl

  • 摘要: 1-脱氧-D-木酮糖-5-磷酸还原异构酶(1-deoxy-D-xylulose-5-phosphate reductoisomerase,DXR)是萜类甲基赤藓醇-4-磷酸途径(methylerythritol-4-phosphate pathway,DXP/MEP)上的第二个限速酶。本研究以5种化学型的香樟(Cinnamomum camphora)作为实验材料,基于转录组数据,运用快速扩增cDNA末端技术(RACE),从香樟中克隆出DXR基因的cDNA,命名为CcDXR1(GenBank登录号: KU886266)。该基因开放阅读框(ORF)长1 413 bp,编码470个氨基酸残基,生物信息学分析推测其分子质量为51.1 kD,等电点(Theoretical pI)为6.62,不含信号肽,不存在跨膜结构。结合蛋白质保守区以及进化树分析,证实该基因确为DXR家族基因。利用实时荧光定量PCR检测得出香樟中CcDXR1基因在成熟叶片表达量高于幼嫩叶片,根与叶中表达量相当,枝中最低。CcDXR1在香樟5种化学型中的表达量不同,在龙脑樟中的表达量要高于油樟、芳樟、脑樟和异樟4种化学型。本研究首次从香樟中克隆出了CcDXR1序列全长,并揭示了CcDXR1在不同组织、生长时期及5种化学型中的差异表达,为香樟萜类化合物生物合成途径关键酶基因的挖掘提供研究基础。

     

    Abstract: 1-Deoxy-D-xylulose-5-phosphate reductoisomerase (DXR) is the second rate-limiting enzyme of terpenoid biosynthesis in the methylerythritol-4-phosphate pathway. According to the transcriptome database of Cinnamomum camphora, the DXR cDNA was cloned by rapid amplification of cDNA ends (RACE) from C. camphora, and was named CcDXR1 (GenBank number: KU886266). The ORF of CcDXR1 is composed of 1 413 bp, and it encodes 470 amino acids. The bioinformatics analysis suggests that the molecular weight of the encoded protein is 51.1 kD and the theoretically isoelectric point is 6.62, and there is no signal peptide and transmembrane structure in putative protein. The analysis of sequence alignment and phylogenetic tree showed that the CcDXR1 belonged to the DXR family. The results of the realtime PCR indicated that expression level of CcDXR1 in mature leaves was higher than tender leaves, which in roots was similar to leaves and the lowest in branches. The camphor is divided into five chemotypes, according to the main chemical compounds in C. camphora. It also showed that the expression level of CcDXR1 in borneol C. camphora was highest than that in cineol, iso-nerolidol, camphor and linalool. Our results revealed that the expression level of CcDXR1 exhibits diversity among plant tissues, growth periods and five chemical types, and the research provides foundation for further study of the terpenoids biosynthetic pathway in C. camphora.

     

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