药学学报, 2020, 55(2): 335-344
谢德金, 叶友杰, 杨德明, 张娅欣, 何天友, 陈礼光, 郑郁善. 巴戟天MoDXR基因及其启动子的克隆与分析[J]. 药学学报, 2020, 55(2): 335-344.
XIE De-jin, YE You-jie, YANG De-ming, ZHANG Ya-xin, HE Tian-you, CHEN Li-guang, ZHENG Yu-shan. Cloning and analysis of the DXR gene and its promoter in Morinda officinalis[J]. Acta Pharmaceutica Sinica, 2020, 55(2): 335-344.

谢德金1, 叶友杰1, 杨德明1, 张娅欣1, 何天友2, 陈礼光1, 郑郁善1,2
1. 福建农林大学林学院, 福建 福州 350002;
2. 福建农林大学园林学院, 福建 福州 350002
从巴戟天中克隆MEP途径中的1-脱氧-D-木酮糖5-磷酸还原异构酶基因MoDXR及其启动子序列并进行生物信息学分析、启动子区顺式作用元件分析,以及进行原核表达分析。根据巴戟天转录组中DXR基因原始序列和NCBI-ORFfinder分析,设计特异性引物,并进行RT-PCR扩增和生物信息学分析;采用染色体步移克隆MoDXR基因的5'端启动子序列;通过亚细胞定位分析MoDXR基因在细胞中的位置;构建原核表达载体pET-28a-MoDXR,导入BL21(DE3)表达感受态细胞后,在IPTG诱导下表达。从巴戟天中克隆的MoDXR基因,其cDNA全长2 015 bp,预测的阅读框大小为1 425 bp,编码474个氨基酸,分子质量为51.27 kDa;BlastP序列比对分析表明MoDXR基因与其他植物的DXR基因具有高度同源性,如:咖啡树DXR(CaDXR)、萝芙木DXR(RvDXR);系统进化发育树分析显示,巴戟天MoDXR蛋白与小粒咖啡和栀子的DXR蛋白聚为一类,其亲缘关系最近;由亚细胞定位可知MoDXR基因所编码的蛋白定位于叶绿体上;MoDXR基因5'端启动子序列长度为1 493 bp,包含了与光响应、胁迫响应和激素响应有关的多种调控元件;SDS-PAGE结果表明pET-28a-MoDXR重组蛋白的大小与预期相符,但是以不容性的包涵体的形式表达。成功克隆了MoDXR基因及其启动子序列并对其进行了生物信息学分析和启动子区顺式作用元件分析;后期需要优化MoDXR原核表达体系,为进一步纯化MoDXR蛋白,研究其结构和功能奠定基础。
关键词:    巴戟天      1-脱氧-D-木酮糖-5-磷酸还原异构酶基因      启动子      生物信息学分析      原核表达     
Cloning and analysis of the DXR gene and its promoter in Morinda officinalis
XIE De-jin1, YE You-jie1, YANG De-ming1, ZHANG Ya-xin1, HE Tian-you2, CHEN Li-guang1, ZHENG Yu-shan1,2
1. College of Forestry, Fujian Agriculture and Forestry University, Fuzhou, 350002, China;
2. College of Landscape, Fujian Agriculture and Forestry University, Fuzhou, 350002, China
The objective of this research was to clone 1-deoxy-D-xylulose 5-phosphate reductoisomerase gene (MoDXR) and its promoter sequence from Morinda officinalis and carry out bioinformatic analysis, cis-acting elements analysis, and prokaryotic expression. On the basis of the MoDXR gene sequence obtained from the M. officinalis transcriptome and with NCBI-ORFfinder analysis, a pair of specific primers were designed, and used for RT-PCR amplification. The promoter region sequence at the 5' end of MoDXR gene was isolated by the genome walking technique. Localization of MoDXR was carried out by subcellular analysis. The prokaryotic expression plasmid pET-28a-MoDXR was constructed and transfected into Escherichia coli BL21(DE3) chemically-competent cells; the recombiant plasmid expressed fusion protein after the induction by IPTG. The full-length cDNA of MoDXR was 2 015 bp, and open reading frame (ORF) size was 1 425 bp, and it encoded 474 amino acid residues and had a molecular mass of 51.27 kD. Sequence comparison with BlastP to the NCBI database revealed that MoDXR had high sequence similarity with many other DXRs, such as Coffea arabica DXR (CaDXR) and Rauvolfia verticillata DXR (RvDXR). A phylogenetic tree revealed that MoDXR had its closest relationship with DXR from Coffea arabica and Gardenia jasminoides. The subcellular localization revealed that MoDXR protein was located on the chloroplast. Plantcare analysis indicated that the promoter region sequence of MoDXR was 1 493 bp, covering multiple light, stress, and hormone-responsive cis-regulatory elements; protein electrophoresis showed that the expressed protein was the anticipated size. This research lays the foundation for further purification and structural and functional characterization of the MoDXR protein.
Key words:    Morinda officinalis    1-deoxy-D-xylulose 5-phosphate reductoisomerase    promoter    bioinformatics analysis    prokaryotic expression   
收稿日期: 2019-09-02
DOI: 10.16438/j.0513-4870.2019-0707
基金项目: 福建省科技创新平台资助(2008Y2001).
通讯作者: 郑郁善,Tel:86-591-83856104,E-mail:zys1960@163.com
Email: zys1960@163.com
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谢德金  在本刊中的所有文章
叶友杰  在本刊中的所有文章
杨德明  在本刊中的所有文章
张娅欣  在本刊中的所有文章
何天友  在本刊中的所有文章
陈礼光  在本刊中的所有文章
郑郁善  在本刊中的所有文章

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