A Preliminary Study On The CDNA Cloning And Function Of Undaria Pinnatifida Suringar Encoding Phytoene Desaturase

Carotenoids are important natural pigments,widely distributed in photosynthetic bacteria,fungi and higher plants.Fucoxanthin,which is one of major carotenoids,accounts for more than 10%of the total carotenoids and has a variety of biological functions.Undaria pinnatifida Suringar,rich in fucoxanthin,has high nutritional values and strong environmental adaptability.However,the fucoxanthin biosynthetic pathway in brown algae is unclear,and there are few reports on the key genes of the synthetic pathway.In this study,the cDNA encoding phytoene desaturase?PDS?,a key enzyme which catalyzes the formation of?-carotene,was obtained by RT-PCR method from brown seaweed U.pinnatifida Suringar.The cDNA of the UpPDS gene was 1707 bp in length including a complete open reading frame and encoding a total of 568 amino acids.Compared with the amino acid sequences of PDS from other species,it was found that phytoene desaturases from different species are highly conserved during evolution and UpPDS protein had a close relationship with that from brown algae such as acid algae PDS and sargassum PDS.The UpPDS was cloned into pET-28a to construct expressing vector pET-28a-UpPDS.The expressing plasmid pET-28a-UpPDS was co-transformed with pACPHYTipi?containing crtB and crtE?into E.coli BL21?DE3?to construct engineering bacteria.The color complementation experiment of engineering E.coli and high performance liquid chromatography?HPLC?method were used to verify the biological activity of the enzyme encoded by UpPDSin engineering bacteria,and results showed that UpPDS protein could catalyze the production of?-carotene from the substrate phytoene.The single-factor conditions were used to analyze the?-carotene production,which included different induction starting OD₆₀₀,different IPTG concentrations and different induction temperatures and different induction time,and then the L₉?3⁴?orthogonal test was used to optimize caroteinoids fermentation conditions of the engineering bacteria.Results showed that?-carotene productiuon in engineering bacteria could reach 5.0 mg/L under optimal conditions.The plant expression vector pCAMBIA2300-UpPDS was constructed and transformed into Agrobacterium LBA4404,and then transferred into tobacco plants by infection.Transgenic tobacco plants were identified by PCR amplification and Southern blotting.Assay of real-time quantitative PCR ananlysis indicated that the expression level of the target gene UpPDS differed greatly in different transgenic tobacco plants.Spectrophotometry was used to determine the carotenoids content in the leaves trangenic plants,and the results showed that the content of carotenoids in the leaves of transgenic plants was higher than that of wild tobacco,and the maximum content increased 1.29times compared with that of wild plants.