番茄八氢番茄红素合成酶基因的克隆及超量表达载体构建

Cloning of Phytoene Synthase cDNA from Tomato and Construction of the Over-expression Vector

八氢番茄红素合成酶是植物类胡萝卜素生物合成途径中促进番茄红素合成的关键酶,根据番茄该酶的编码基因序列设计一对引物,通过RT-PCR在番茄中扩增出一个约1500 bp的全长cDNA片段。测序结果表明,此片段包含一个长为1239 bp的完整的编码框,其氨基酸序列与辣椒、向日葵、万寿菊、枸杞、番木瓜、温州蜜柑、拟南芥八氢番茄红素合成酶基因编码的氨基酸序列的一致率分别为88%、75%、75%、74%、73%、73%、71%。对这个全长片段构建了超量表达载体,酶切检测表明该片段已插入植物表达载体。

Phytoene synthase （PSY） is a key enzyme in the carotenoid biosynthetic pathway. A 1500 bp full-length cDNA fragment was amplified by RT-PCR from tomato using a pair of specific primers based on the coding sequence of PSY. This fragment contained a complete ORF of 1239 bp. The deduced amino acid showed 88%, 75%, 75%, 74%, 73%, 73% and 71% homology with PSY from hot pepper, sunflower, marigold, medlar, papaya, satsuma mandarin and Arabidopsis thaliana, respectively. Using the amplified fragment, the over-expression vector was constructed and identified by digestion with appropriate enzymes.