Clone ZDS Gene From Tomato And Construct Vector For Expressing Lycopene In Rhodobacter Sphaeroides Mutant TC40

Lycopene, one of the carotenoid with powerful character of Anti-oxidation, is a bright red carotenoid pigment, a photochemical found in tomatoes and other red fruits. Lycopene is a proven antioxidant. Antioxidants neutralize free radicals, which may damage the body's cells. Now the researches on lycopene biosynthesis and the regulation mechanism have received considerable attention in molecular biology. In tomato, a model of the carotenoid pathway as following has been proposed: IPP→DMAPP→GPP→FPP→GGPP→Phytone→Lycopene.The Lycopene located at the most downstream position of the carotenoid pathway in plants. The conversion of GGPP to phytoene is the first committed step in carotenoid biosynthesis, catalyzed by the enzyme phytoene syntheses. The next two enzymes (phytoene andζ-carotene desaturases) desaturate phytoene andζ-carotene, respectively. The product ofζ-carotene desaturase is lycopene, which is responsible for the characteristic red color of ripe tomato fruit. Phytoene desaturases (CrtI) occurring in nature convert phytoene to either neurosporene or lycopene in most eubacteria. Approximately 10% of known phytoene desaturases, as in Rhodobacter, produce neurosporene, whereas the rest produce lycopene.carotenoid pathway existed in both plants and eubacteria, Here used site-directed evolution introducing tomato ZDS gene into R. sphaeroides to change the product of crtI gene product, a neurosporene-producing enzyme, to lycopene. So it is significant to identify the function of Crt gene, in order to improving genetic manipulation in both plants and eubacteria.So far, in plant bioengineer , CrtI from the bacterium Erwinia uredovora has been overexpressed in tomato. Fruit-specific expression was achieved by using the tomato polygalacturonase promoter. Study on ZDS will make us better understand the mechanism of Crt genes expression. In this research, the crtI gene of tomato was heterologously expressed in the R.sphaeroides mutant bacteria TC40, endowing TC40 with property to producing lycopene after transformation. The main results change the product of crtI gene products are listed bellow.①Extract total RNA from fruit, flower, leaf and stem of wild-type tomato(Lycopersicon esculentum) separately②A full-length cDNA, named ZDS, was cloned from fruit ,flower and leaf of wild-type tomato by RT-PCR and PCR. The length of ZDS cDNA was 1766 bp encoding a polypeptide of 589 amino acids Comparison of this protein sequence with Capsicum and Arabidopsis showed that the amino acid homology of both was 79.9%③ZDS gene was inserted into plasmid pMD18-T, named pMD18-ZDS.④WYG gene was inserted into plasmid pMD18-T, named pMD18-WYG. The plasmid pMD18-WYG digested by the SacI, XbaI, gain the fragment of WYG insert into vector pRKR5 cut with SacI and XbaI, named pRKR5-WYG.⑤The plasmid pMD18-ZDS digested by the XhoI, gain the fragment of ZDS ,linking with the plasmid pRKR5-WYG digested by the XhoI, obtain the overexpress vector pRKR5-ZDS.⑥Use DNA Conjugation method, transfer pRKR5-ZDS into TC40.⑦Overexpress pRKR5-ZDS in TC40.