Cloning And Prokaryotic Expression Of Phytoene Synthase Gene (PSY1) In Wheat (Triticum Aestivum)

Carotenoids, which can be divided into carotenes and its oxidative derivatives (xanthophylls), are one large group of natural products that are mostly synthesized from isoprene units. They can be biosynthesized in many kinds of bacterium, fungi and plants and many of them are closely related to human health due to their special physiological functions. The nutritional value will be evidently promoted by improving carotenoid contents through building carotenoid synthasizing pathway in wheat.A pair of RT-PCR primers were designed and synthesized based on the published gene sequence of wheat PSY1. About 1400 bp target DNA sequence were amplified by reverse transcription polymerase chain reaction depending on the template of total RNA isolated from wheat leaves, and cloned to pMD18-T simple vector, transformed into E.coli competent cells. The result showed that the length of the acquired clone was 1436 bp, containing a complete Open Reading Frame of 1287 bp which encode a protein of 428 amino acids. We found that the clone had high homology with other species's PSY1 gene through cladogram analysis,and confirmed it was the wheat PSY1 .By the technology of DNA recombination, the wheat PSY1 clone was subcloned to BamHI/SalI sites of the expression plasmid pET-32a vector. The recombined plasmid, named pET-32a-PSY, was transformed into E.coli BL21 competent cells and induced with IPTG. SDS-PAGE illuminated that about 47 kD protein was expressed in the induced recombinant E.coli BL21, with nothing in the control group.In conclusion, we successfully cloned wheat PSY1 and constructed the recombinant pET-32a-PSY which was expressed in prokaryotic E.coli BL21 cells. All underlied the future research on wheat phytoene synthase gene.