The Research Of Cloning Of RS Gene In Grape And Its Genetic Transformation Into Astragalus

Resveratrol Synthase(RS)is a limiting enzyme in the biosynthetic pathway of resveratrol(Res).RS gene,whether in plants,or in micro-organisms have achieved genetic transformation and production of active products,and Res have biologic activity in terms of biosynthetic metabolic pathways and expression regulation in two expression systems.With the increasing demand for Res,direct extraction of active ingredients,has been far from being able to meet the demand for Res.The production of the active ingredient is improved by plant improvement or genes overexpression through Modern biotechnology and genetic engineering.Therefore,the utilization of transgenic plant technology for heterogeneous plant production Res research prospects has a broad research prospects.In this study,RS gene was cloned by genetic engineering technique and bioinformatics analysis was carried out,and then RS gene,which cloned from the grape,was transferred into astragalus by Agrobacterium-mediated method,As the main active ingredients of Astragalus flavonoids and resveratrol biosynthetic pathways are from the phenylalanine synthesis pathway,they have the same synthetic process,So Astragalus was chose as receptor material,Not only can explore the RS gene transformation of heterologous plant Astragalus,to achieve the possibility of heterotrophic production of resveratrol,and provide technical support for astragalus transgenic,but also can improve the resistance of Astragalus,and support the function of resveratrol synthase,which provides a valuable theoretical reference for the modernization of Chinese medicine.The results of this study are as follows:1.The RS gene encodes a protein containing 310 amino acid residues,the gene encodes resveratrol synthase by the analysis by Blast2 go software.2.The expression vector of RS gene was successfully constructed: p BR121-RS.And the genetic transformation of astragalus explants was carried out by Agrobacterium tumefaciens genetic transformation.A total of 204 resistant trees were obtained,and 11 transgenic positive astragalus plants were obtained after rooting culture,cultivation and transplanting.3.Finally,three transgenic Astragalus positive plants proved that the RS gene was fully integrated into the Astragalus genome and expressed to produce resveratrol by DNA,Southern blot and RT-PCR detection.4.Three strains with Southern blot hybridization were found to have higher resveratrol content than non-transgenic plants by comparing the content of resveratrol in transgenic and non-transgenic plants.The resveratrol content in non-transgenic plants is 2029.46? g/g,but the resveratrol content in 3 transgenic plants is 2998.32? g/g,3854.66? g/g,3989.77? g/g,respectively,increasing48.23%?91.27% and 96.59%,respectively.The regeneration and transformation system of Astragalus membranaceus were established in this study,and the RS gene derived from Kyoho grape was successfully transferred into astragalus for expression,Astragalus transgenic positive plants were obtained.This can not only explore the possibility of RS gene transformation of heterologous plant Astragalus,to achieve the production of resveratrol by heterogeneous plants,but also provide technical support for transgenic breeding of astragalus;It also can improve the resistance of Astragalus,and provide support for the function of resveratrol synthase,and it also provides a valuable theoretical reference for the modernization of Chinese medicine.