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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。
Purpose: The fruits of Gardenia jasminoides are rich in crocetin and crocin from the oxidative cleavage of a carotenoid-zeaxanthin. In this paper, the gene of phytoene synthase (PSY), a key enzyme in the pathogen of gardenia carotenoid biosynthesis, was cloned and the mechanism of the synthesis of crocin and crocetin in gardenia fruit was preliminarily studied. Methods: The full length cDNA of GjPSY was cloned from the cDNA library of gardenia fruit. The GjPSY cDNA was transformed into E. coli BL21 (DE3) by inserting the cDNA of GjPSY into the expression vector pET-21b. The recombinant GjPSY protein was induced by IPTG and the protein was purified by nickel affinity chromatography. RESULTS: The GjPSY cDNA was 1 876 bp in length and contained an open reading frame of 1 314 bp. The predicted GjPSY cDNA was 437 amino acids in length. The recombinant GjPSY protein was expressed and purified in E. coli. CONCLUSION: The similarity of the GjPSY protein sequence with other plant PSY sequences is between 55 and 93%. The predicted GjPSY spatial structure has the conserved structural features of PSY protein. The constructed expression vector can be used for the functional study of GjPSY.