Cloning And Prokaryotic Expression Of Resveratrol Synthase Gene Of Peanut

Resveratrol (hydroxystilbene) is a phytoalexin which was detected in several plant families. It was revealed that resveratrol played an important role of anticancer and prevention from cardiovascular disease. Since resveratrol synthase(RS) is the key enzyme in the synthesis of resveratrol, the research about the synthase and its gene is of great significance. It not only will shed light on the catalytic mechanism of the synthase but also contribute to the activity of transferring this gene to plants to improve their ability of resistance to disease.The purpose of the experiment is to clone the resveratrol synthase gene from peanut and express the gene in E. coli. It will lay a foundation for further research about the functions of the synthase and genetic transformation to plants.In the experiment, the method of RT-PCR(reverse transcription-polymerase chain reaction) was used to obtain the target gene. After mRNA was isolated from the peanut and transcripted to cDNA, the gene of interest was amplified with the RS- gene- specific primers. Then the gene was inserted to T-vector (pMD-18T) . Sequence analysis indicated that the gene is consisted of 1. 2kb and encode 390aa. The identity of the derived protein with several peanut resveratrol synthases registered in Genbank is 93%-96%.The gene was sub-clone to prokaryotic expression vector pQE30a and the recombinant plasmid was transformed to E. coli M15.Sequencing the construct showed that both of the sequence and the reading frame were correct. The gene was expressed by induction of IPTG and the expression product accounted for 24% of the total proteins. After sonicating the bacterial, soluble proteins was collected and purified with His-affinity column. A single band was shown in the SDS-PAGE analysis.In summary, the resveratrol synthase gene was successfully cloned from the peanut and expressed in E. coli.