Gene Cloning And Prokaryotic Expression Of RS Of GRAPE

The purpose of the experiment is to clone the resveratrol synthase gene from Vitis pesudoreticulata Baihe and express the gene in E.coli BL21. So we can construct a new functionality gene-project bacillus.Resveratrol synthase(RS) is the key enzyme in the synthesis of resveratrol. Resveratrol (hydroxystilbene) is a phytoalexin which was detected in several plant families. It was revealed that resveratrol played an important role in resisting oxygenation,anti-bacterium and preventing from inflammation. The research about the synthase and its gene is of great important for it can make us utilize the resveratrol efficiently.In the experiment, the method of RT-PCR(reverse transcription-polymerase chain reaction)was used to obtain the target gene. After total RNA was isolated from the grape leaves and transcripted to cDNA, the gene of interest was amplified with the cDNA template and RS gene primers. Then the gene of interest was amplified again with RS-gene-specific primers which has enzyme digestion locations.The gene was inserted to pGEM-T Easy vector after it was recycled and purifided to constract the reconstraction plasmid pGEM-T-RS. Then the new plasmid was transformed into E.coli DH5α. Sequence analysis indicated that the gene is consisted of 1179bp and encoded 392aa. The identity of the gene with several grape resveratrol synthases registered in Genbank was 96%-98% and the derived protein was 97%-99%.Then the gene was digested from pGEM-T-RS and was sub-clone with prokaryotic expression vector pET-22b(+) to constract the reconstraction plasmid pET-RS. The recombinant plasmid was transformed into E.co1i BL21. We conformed we had successfully constructed the expression vector through antibiotic screen and specific enzyme digestion.Cultivating reconstraction bacterium and finding their growth curve by turbidimetry. We found the bacterium were in logarithmic phase after 4 hours. So the gene was expressed by inducing of IPTG after cultivated 4 hours. After sonicating the bacterium, the upper solution was collected. And a certain band was shown in the SDS-PAGE analysis.In summary,the resveratrol synthase gene was successfully cloned from the Vitis pesudoreticulata Baihe and expressed in E.coli BL21 correctly. It established the foundation for using the bacterium to producing probiotics.