Enrichment Of Four Kinds Of C-glycosides Flavones For Diabetes In Tomato Fruits

Diabetes is a serious metabolic disease characterized by hyperglycemia.Although there are many drugs that can treat diabetes at present,its price,sequelae,convenience,etc.have always been factors worth considering.Therefore,other convenient ways to treat or control the process of diabetes will eventually become the mainstream to guide the market.Many studies on diabetes have shown that C-glycosides flavones are excellent in the treatment of diabetes.Therefore,if C-glycosides flavones can be expressed in tomato fruit,eating tomato can play a role in reducing sugar.Many studies have confirmed that vitexin,isovitexin,orientin and isoorientin,four highly active secondary metabolites of C-glycosides flavones in plants,have obvious anti diabetes effects.Objective: The flavanone 2-hydroxylase(CYP93G2)and 2-hydroxyflavanone C-glycosyltransferase(OsCGT)genes from rice(9311 and Japonica rice)play an important role in the biosynthesis of vitexin,isovitexin,orientin and isoorientin,and use gene engineering technology to specifically enrich four kinds of C-glycosides flavones in tomato fruits to obtain edible tomatoes containing effective anti-diabetes ingredients,It will help many patients with diabetes and the development of related drugs.Methods: In this study,based on the principle of synthetic biology,using the fruit specific promoter p2A11,a plant expression vector was constructed to start the CYP93G2 gene encoded by Os06g01250,the gene on OsCGT encoded by LOC9266253 and the fusion gene of the two,and the tomato was processed through agrobacterium-mediated genetic transformation;The transformed plants were identified by RTq PCR and other techniques,and the content of tomato fruit components was analyzed by HPLCResults:(1)The fruit specific promoter p2A11 from the total DNA of tomato plants was successfully cloned,with a total length of 1813 bp,and was used to replace the 35 S promoter sequence of p CAMBIA2300vector;(2)The Os06g01250 gene was successfully cloned from the leaf c DNA of 9311.The total length of the gene was 2157 bp,and the total length of the CDS region was 1687 bp(containing a 130 bp intron sequence,using fusion primers).The p CAMBIA2300-p2A11-CYP93G2-OCS vector was successfully constructed,and obtained tomato plants with high CYP93G2 expression using Agrobacterium tumefaciens infection method;(3)The LOC9266253 gene was successfully cloned from 9311 leaf c DNA,with a full length of 1614 bp.p CAMBIA2300-p2A11-OsCGT-OCS vector was successfully constructed,and obtained tomato plants with high OsCGT expression by Agrobacterium infection;(4)The p CAMBIA2300-p2A11-CYP93G2-T2A-OsCGT-OCS fusion expression vector,which contains 3036 bp of full length CYP93G2-T2 AOsCGT,was constructed using CYP93G2 and OsCGT.Tomato plants simultaneously expressing CYP93G2 and OsCGT genes were obtained by infection of tomato calli with Agrobacterium tumefaciens;(5)The components of the transgenic tomato fruit were determined by HPLC.The results showed that the transgenic tomato fruit contained four effective components of C-glycosides flavones,namely vitexin,isovitexin,orientin and isoorientin;Conclusion: In this study,by expressing CYP93G2 and OsCGT genes in tomato fruits,the four Cglycosides flavones metabolic pathways were completely opened in tomato,and tomato fruits expressing functional components for improving diabetes mellitus were obtained.This study provides theoretical and practical evidence for further exploration of this metabolic pathway.