Resveratrol Biosynthesis In Transgenic Hairy Roots Of Mulberry

Resveratrol is a non-flavonoid polyphenol compound containing stilbene structure synthesized by plants under stress.It not only plays an important role in the process of plants responding to stress,but also is a natural antioxidant with a variety of physiological and pharmacological activities,which has a wide range of applications in the fields of medicine,food and cosmetics.Although resveratrol has been found in more than 100 plants,the content of resveratrol in natural plants is generally low,and only a few plants with relatively high content of resveratrol have development and utilization value.However,there are the problems with the extraction and utilization of resveratrol such as high extraction cost,low yield,shortage of raw materials,etc.Therefore,improving the ability of plants to synthesize resveratrol by genetic engineering technology and utilizing plant biosynthesis to synthesize resveratrol are effective approaches and feasible strategies to solve the problems faced by the development and utilization of resveratrol.Mulberry is not only rich in resveratrol in root,stem,leaf and fruit,but also has high growth speed and high biological yield,making it an ideal species for resveratrol biosynthesis.However,no research report has been made on the biosynthesis of resveratrol by mulberry.In this study,STS/CHS gene family members were cloned from‘Dashi’,and the resveratrol synthase gene was identified by using tobacco transient expression system.The resveratrol synthesis ability of the resveratrol synthase gene was verified by recombinant Escherichia coli.Moreover,the technical system for biosynthesis of resveratrol by mulberry transgenic hairy root system was established.The results given could lay a foundation for efficient biosynthesis of resveratrol using hairy roots of mulberry,and also provide a reference for biosynthesis of other secondary metabolites using mulberry,which was of great significance for the diversified development and utilization of mulberry and the development of mulberry industry.The main results of this study are as follows:(1)Cloning and sequence analysis of the STS/CHS genes.Based on the transcriptome sequence information of Mori Fructus from big ten,seven member genes of STS/CHS gene family were cloned from ‘Dashi’,and the encoded proteins showed high homology,and the amino acid sequence consistency was up to 85.75%,and all of them contain the characteristic signal region and active center region of STS/CHS gene family.Phylogenetic tree analysis showed that the seven members of the STS/CHS family could be significantly divided into two evolutionary branches,whose biological functions might be different.(2)Identification of resveratrol synthase gene by tobacco transient expression system.The tobacco was infected by the agrobacterium-mediated transient infection technology,and the seven members of the STS/CHS family were transiently expressed in the leaves of tobacco.The resveratrol content measurement results showed that the transient expression of the seven members of the STS/CHS gene family in tobacco leaves could improve the resveratrol content in the leaves.Among them,the leaves transfected with MbSTS7 and MbCHS3 genes had the highest resveratrol content,indicating that the seven members of the STS/CHS gene family were involved in the biosynthesis of resveratrol.However,the MbSTS7 and MbCHS3 might be the main resveratrol synthase genes.(3)Recombinant E.coli was used to verify the resveratrol synthesis ability of MbSTS7 and MbCHS3 genes.The fusion gene prokaryotic expression vector was successfully constructed by connecting MbSTS7 and MbCHS3 genes with 4-coumaroyl-Co A ligase gene(4CL)through amino acid linker(GSG),respectively,and transferred to E.coli BL21(DE3).Through liquid fermentation culture,the yields of 0.4 mg/L and 1.3 mg/L resveratrol were obtained in the fermentation broth of the Mb4CL-STS7 transgenic or Mb4CL-CHS3 transgenic fusion gene strain,respectively.The results further proved that the MbSTS7 and MbCHS3 were resveratrol synthase genes.(4)Producing resveratrol by mulberry transgenic hairy roots.p ROKⅡ plant expression vectors of MbSTS7 and MbCHS3 genes were constructed,respectively.The hairy roots of mulberry transgenic MbSTS7 and MbCHS3 genes were successfully obtained using the root transformation system mediated by Agrobacterium rhizogenes K599.The optimal extraction process of resveratrol from mulberry hairy roots was as follows: the ratio of solid to liquid is1:8,extraction temperature is 55℃,extraction time is 60 min,and ultrasonic power is 100 W.The resveratrol contents in the transgenic hairy roots of mulberry with transgenic MbSTS7 and transgenic MbCHS3 gene were 0.12 mg/g and 0.13 mg/g,respectively,which were significantly higher than those in the wild-type and transgenic empty hairy roots.