Investigation Of Resveratrol Preparation By Plant Bio-reactor--Isolation Of Peanut Resveratrol Sythesis Enzyme 1 (PNRS1) And Its Functional Anaysis In Plants

Plant bio-reactor means to use transgenic plants to produce biological agents, such as recombinant pharmaceutical proteins, industrial enzymes, lipids and secondary metabolites et. al, by gene engineering method with the advantages such as enconomy, high efficiency and safty. It could provide virtually unlimited quantities of proteins to meet the increased requirments for both human health care and the bioscience research.Resveratrol (Res) is a naturally occurring stilbene, a kind of polyphenolic compounds, found in a limited number of unrelated plant species. It has been considered as a phytoalexin in plants, and many studies have also shown it has health benefits such as antioxidant activities, cancer prevention, blood thinning, life span extension et al. As the content of Res is low in plant, it is very difficulty and costly to be extracted plentifully by traditionally method. Resveratrol synthase (RS) is the key and necessary enzyme in Res synthesis procedure, and investigation of RS could shed light on study of Res synthesis pathway.In this paper, the RS gene of peanut (PNRS1) is coloned by the method of molecular biology, and it is expressed by prokaryotic system and geneticaly transformed into plants such as tobacco.The following progresses have been made by now:A RS cDNA named PNRS1 (accession No. FM955393) was cloned according to RT-PCR (reverse transcription-polymerase chain reaction) protocol using root cDNA of peanut as template. Sequence alignment showed that the homology of PNRS1cDNA sequence is up to 95% with that of AY170347, and the similarity of its amino acid sequence is up to 96% with that of AAO11837. The feature structure and activity site are all present in the sequence. RT-PCR analysis indicated that PNRS1 was specically expressed in root of peanut, and could be induced under UV irradiated conditions.PNRS1 was sub-cloned into prokaryotic expression vector pET-28a. After the gene sequence and reading fram were confirmed correctly by sequencing, the recombinant plasmid named pET-28a-PNRS1 was then transformed into E. coli BL21(DE3). The IPTG induced expresstion product was determined by SDS-PAG and showed a band of with the molecular weight about 46 KD. The further analysis also show that the PNRS1 gene could be efficiently induced when using 1 mmol/L IPTG to induced for 4 h after the transformants were cultured for 3 h at 37℃.Meantime, the cDNA of PNRS1 was confirmed correctly by sequencing and integrated into the multiple cloning site (MCS) of plant expression vectors, such as pBI121 to get the construct pBRS, and a modified p1301 vector, which has CaMV35S as promoter with NOS as terminator to get the construct p1301-35s-RS1. The constructions were then transformed into LBA4404, and the genetic transformation into plants, such as tabacoo leaf, was carried out by Agrobacterium–mediated method. After the transgenic plants were generated and verificated, parts of transgenic lines were examined by the method of UV spectrophotometry and HPLC to analysis the concentration of Res. Compared with that of control, the Res content were all raised more or less in the transgenic plants, which suggested the heterologous gene integrated in the genomic DNA of the plants and play role in Res sysethsis pathway.In summary, the successful cloning of the peanut gene PNRS1 and effective expressing of it in prokaryotic expression system and plant laid the foundation for further investigation on its biological function and shed light for the improvement of disease resistance and health caring quality of crops and food.