南方红豆杉DXS基因的克隆与功能分析

Molecular cloning and characterization of 1-deoxy-D-xylulose 5-phosphate synthase gene from Taxus chinensis

目的从南方红豆杉Taxus chinensis获得紫杉醇生物合成途径关键酶1-脱氧-D-木糖醇-5-磷酸合成酶(1-deoxy-D-xylulose 5-phosphate synthase,DXS)基因(TcDXS)并进行生物信息学和表达模式分析以及亚细胞定位和功能鉴定。方法采用RACE技术获得TcDXS全长;利用qRT-PCR分析TcDXS在南方红豆杉不同部位的表达情况;采用亚细胞定位分析TcDXS在细胞中的位置;用颜色功能互补实验检测TcDXS功能。结果获得的TcDXS由3 031个核苷酸组成,包含了2 229 bp编码区,编码742个氨基酸,其蛋白相对分子质量为79 400,等电点(p I)为7.99;qRT-PCR检测表明TcDXS在南方红豆杉的嫩叶柄中表达量最高,其次是叶和皮,根和茎中的表达量较低;亚细胞定位结果表明,TcDXS定位在叶绿体中;功能互补实验结果表明,在共同转化p AC-BETA和pTrc-TcDXS的大肠杆菌菌落呈现出橘黄色。结论 TcDXS的克隆和功能分析为深入研究紫杉醇生物合成途径和实现其代谢工程奠定了基础。

Objective To obatin the key enzymes of 1-deoxy-D-xylulose 5-phosphate synthase（DXS） in taxol biosynthetic pathway from Taxus chinensis（TcDXS）, and carry out the bioinformatics analysis, tissue profile, subcellular localization, and functional complementation assay. Methods RACE technologies were used to obtain the full length c DNA of TcDXS for the bioinformatics analysis. Semi-quantitative PCR was used to detect the gene expression levels in different parts of T. chinensis. The localization and function of TcDXS were carried out by subcellular localization and functional complementation assay. Results The full-length c DNA of TcDXS was 3 031 bp containing a coding sequence of 2 229 bp encoding a 742-amino-acid residues which was predicted to have a molecular weight of 79 400 and an isoelectric point of 7.99. The qRT-PCR results showed that the highest expression level of TcDXS was detected in petioles and followed by leaves and barks. However, the expression of TcDXS was very low in roots and stems. What＇s more, functional complementation assay results showed that the E. coli, co-transformed with PAC-BETA and pTrc TcDXS, was changed to orange. Conclusion TcDXS was considered to play an essential role in the control of taxol biosynthesis and provided a target for the metabolic engineering of taxol production and plant molecular breeding in T. chinensis.