Tomato Transformed With A Vitis Stilbene Synthase Gene Produce Resveratrol

Resveratrol(trans-3,5,4 -trihydroxystilbene) is a naturally occurring phytoalexin produced by some spermatophytes,such as grapevines,in response to injury. Research shows that resveratrol possesses activities of modulation of lipid metabolism,inhibition of lipid peroxidation(LDL,membranes) and platelet aggregation,etc. Meanwhile as it can reduce coronary heart-disease mortality and features the activity of anti-inflammatory,it is condidered to have some beneficial effects on human health. As a popular vegetable,tomato is rich in vatamin A,B,P and other nutrients;among them,lyxopene can prevent prostate gland tumor. So introducing stilbene synthase gene into tomato to get new health-protection tomato will have important social and economic effect.Our research is made up of four parts;the cloning and sequencing of stilbene synthase;tomato genetic transforming mediated by Agrobacterium-LBA4404;quantification of resveratrol in transgenic tomato by HPLC analysis.Stilbene synthase genes(l,2)from Vitis venefera were cloned and sequenced. The results of sequence analysis showed the total length of S2 is 1617bp,the homology between S2 and pVIN(reported) is 99%;the total length of SI is 1622bp,the homology between SI and pVIN(reported) is 98%. Deduced amino acid sequence of S1,S2,pVIN were also highly homologous each other(98%,99% in each case). The stilbene synthase genes were excised from the plasmids by BamH I and Sac I digestion and intergrated into a binary vector,pBI121 and pEV2,from which the P -glucuronidase(GUS) gene sequence had been removed by the same digestion to prepare a 35s promoter-stilbene synthase 2-nopaline synthase polyadenylation site construct and a TFP2 promoter-stilbene synthase 1-nopaline synthase polyadenylation site construct. The recombinant plasmids were called pBS2,pEV2S 1 .respectively.There was no evident difference between the cultures of variety TX0014 and anti-Pseudomonas 3 for their sensitivity to Kanamycin(Kan). The leaf discs turned yellow and calli were hardly induced at 50mg/L Kan concentration in the medium M1. Kanamycin at 50mg/L concentration suppressed the root growth and the plantgrowth The efficiency of callus induction and regeneration on the medium Ml containing 50mg/L Kan was the highest when the leaf discs were infected with Agrobacterium tumefaciens containing lOOmol/L AS between 2 and 3 minutes.Five plants (TX0014) regenetated from selection medium were obtained. The .presence of the stilbenesynthase gene 2 in the regenerated plants was checked by PCR . All of the plants showed a clear band corresponding to the relevant sequence of the gene,but the control showed no band. Southern blot analysis suggest that S2 gene has been integrated in the genome of three PCR-positive regenerated plants. The rest two regenerated plants did not show a band corresponding to S2. A HPLC analysis of leaf extract from 3 transformant and 2 non-transformant was made. It indicates that resveratrol was produced in these three transformants at a concentration of 12.5ug/g,4.55ug/g,5.35 ng/g respectively,the resverarol production was not examined in non-transformants.Our research can lay a good foundation for tomato quality breeding,meanwhile the tomato containing resveratrol is also potential for providing protection on human health.