【摘要】 目的:栀子(Gardenia jasminoides)的果实富含由一种类胡萝卜素—玉米黄素裂解氧化转变而来的藏花酸和藏花素。该文通过克隆栀子类胡萝卜素生物合成途径的关键酶—八氢番茄红素合成酶(PSY)的基因,初步研究栀子果实中藏花素和藏花酸的合成机理。方法:从栀子果实的cDNA文库中克隆了GjPSY的全长cDNA。通过将GjPSY的cDNA插入表达载体pET-21b中,转化大肠杆菌BL21(DE3),经IPTG诱导表达重组GjPSY蛋白,用镍亲和层析法纯化该蛋白。结果:GjPSY cDNA全长1 876bp,含有一个长1 314 bp的开放阅读框架,预测编码含437个氨基酸的蛋白质,GjPSY重组蛋白在大肠杆菌中被表达和纯化。结论:GjPSY蛋白序列与其它植物PSY序列的相似性在55～93%之间,预测的GjPSY的空间结构具有PSY蛋白的保守结构特点。所构建表达载体可用于GjPSY的功能研究。更多还原

【Abstract】 Objective: The fruit of Gardenia jasminoides produce and accumulate of crocin,which is derived from zeaxanthin.We investigated the molecular mechanism involved in the synthesis of crocin by cloning the phytoene synthase(PSY) in fruit of G.jasminoides,the PSY is the first committed step in carotenogenesis.Method: The full-length cDNA encoding putative phytoene synthase(GjPSY) was cloned from G.jasminoides fruit cDNA library.The cDNA encoding GjPSY was cloned into the expression vector pET-21b.The recombinant protein was expressed in E.coli BL21(DE3) cells and subsequently purified using Ni2 +-affinity chromatography.Result: The GjPSY cDNA is 1876 bp,contains a predicted 1314 bp open reading frame that encodes 437 amino acids.Specific band of the purified fusion protein appeared at about 50.1kDa was detected by SDS-PAGE.Conclusion: GjPSY protein share 55% to 93% amino acid identity with phytoene synthases from other plants.The 3D model structure of GjPSY revealed typical PSYs tertiary structures.The recombinant vector can be used for GjPSY function assays.更多还原