Expression Of Arachis Hypogasa Resveratrol Synthase Gene In Tabacum Roots

Resveratrol (Res), chemical name 3,5,4’-trihydroxysitlbene, also known as stilbene triphenol is an important phytoalexin that widely exists in Polygonum cuspidatum (giant knotweed), Vitis vinifera (grape), Arachis hypogasa (peanut) and other plants, and is particularly rich in the grape skins and peanut roots. It has a variety of biological activity. It is a natural antioxidant and can reduce blood lipids, inhibit platelet aggregation, anti-cancer, anti-inflammatory, anti-radiation, anti-aging, prevent cardiovascular disease and so on. It is known as the green anti-cancer drugs together with paclitaxel. Because of its deficiency supply from plants in nature, the use of biotechnology is expected to produce resveratrol abundantly. Resveratrol synthase (RS) is the key enzyme of resveratrol biosynthesis. In this study, we used tabacum root-specific expression promoter to drive the expression of peanut resveratrol synthase gene and to produce resveratrol in tabacum root. Results are as follows:1. Extracted resveratrol from root, stem, leaf and hypocotyl of peanut cultivar Minhua6 at trilobite period by solvent extraction, and detected the resveratrol content by RP-HPLC, it was found that the resveratrol content is different with kinds of organs, and the content trend is root> leaf> stem> hypocotyl. Hypocotyl seemed no resveratrol in peanut.2. Primers were designed based on 1503bp NtR12 promoter sequence isolated by our laboratory and the fragments of NtR12 promoter was cloned by PCR from tabacum genomic DNA. After sequencing, five different sequences of the promoter were got. Then, took the advantage of the PLACE, the database available at http://www.dna.affrc.go.jp/PLACE/signalscan.html (Higoetal.1999), to analyze the promoter sequences and it suggests that the sequence with cis-element such as TATA-box, CAAT-box, ROOTMOTIFTAPOX1, and with the characteristics of typical root-specific expression promoter.3. Built the tabacum root-specific (root and stem specific) expression vector pBI121-NtR12-GUSA, pBI121-NtR12-RS, and transformed them and other two expression vectors pBI121-NtR2-RS and pBI121-NtR6-RS into the tobacco cultivar CB-1 and Nicotiana benthamiana by Agrobacterium tumefacien-mediated transformation, we got the transgenic positive plants.4. Agrobacterium rhizogenes 001 and Agrobacterium rhizogenes 002 were used to infect the CB-1 and Nicotiana benthamiana for optimizing the root induction, and it was found that tobacco CB-1 infected by Agrobacterium rhizogenes 001 and Nicotiana benthamiana infected by Agrobacterium rhizogenes 002 to obtain the abundant roots are the optimal choice. Hair roots induced by Agrobacterium rhizogenes vary from strains and plant hosts. The natural rooting and Agrobacterium rhizogenes induced rooting cultures were compared and it was found that the roots growth rate of latter method was much faster than the former.5. The hairy roots induced by Agrobacterium rhizogenes-infection were cultured in liquid medium, and the liquid culture could realize the mass production of roots and its growth rate is much faster than the solid culture has been found.6. After PCR and RT-PCR analysis, we have got many transgenic plants. Assays of AhRS1 expression and resveratrol content in roots, stems and leaves are still in progress to validate the specific expression; and also in roots induced by Agrobacterium rhizogenes for the validation of AhRS1 expression and resveratrol production.