Cloning,Analysis And Functional Verification Of GGP Promoter Of Rosa Roxburghii Tratt

Cili(Rosa roxburghii Tratt)has brought about great interest worldwide due to its extremely high content of L-ascorbic acid(vitamin C,AsA)in fruit.Our previous research showed that the L-galactose pathway may be the main pathway for AsA biosynthesis and GDP-L-galactose phosphatase(GGP)involved in this pathway may be its key enzyme(key genes)in Rosa roxburghii Tratt.In this study,the 'Ginong 5' was used as the material,and the full-length gDNA sequence and promoter sequence of GGP gene were cloned by chromosome walking.The structural features and original components were analyzed about this.We measured the activity of each(deletion)element by a dual luciferase reporter gene assay system;On this basis,responsive to the expression of this gene was verified by the illumination,temperature and some exogenous hormones.The main results obtained in this study were as follows:1.The gDNA sequence of the GGP gene was cloned from the fruit of Rosa roxburghii by PCR technique.Its full-length sequence was 2103 bp,including 4 exons and 3 introns.The intron was a total of 304 bp,between1007-1092 bp,1277-1404 bp,and 1652-1744 bp respectively.The sequence of GGP gene promoter was cloned by chromosome walking method and its length was 2133 bp.The results of promoter cis-acting elements online analysis website showed that GGP gene promoter sequences mainly include: cis-acting elements related to light response,stress response elements,hormone response elements,and tissue specific elements.2.The transient expression vector was constructed and tobacco leaf was used as the host material.The promoter activity was detected by Dual-Luciferase reporter gene detection system.The results showed that the activity of the GGP gene promoter was much higher than that of the deletion body.It was speculated that there may be an enhancer region between-1949 and-989,and this 140 bp played an important role in the activity of the promoter.In addition,experimental studies of promoter deletions suggested that there may be negative regulatory elements between-1220 and-1386,resulting in a sudden increase in the initial activity of the defective QSF3.Light and high temperature could induce the activity of GGP gene promoter.According to the response of the GGP gene promoter deletions to different treatments,it was speculated that CAG-motif,GA-motify and G-Box might play an extremely important role in the light-dark response;The high-temperature response element HSE had a positive effect on the initiation of the promoter.The regulatory effect might also contain unknown temperature regulatory elements;TGACG-motif played an important role in the response of the GGP gene promoter to MeJA.3.Responsive element verification experiments showed that GGP gene promoter activit y was induced by light treatment.The light treatment increased the expression of GGP gene,and then increased the AsA content of Rosa roxburghii fruit.However,the dark treatment inhibited the activity of GGP gene promoter,which resulted in the down-regulation of GGP gene expression and the sharp decrease of AsA content in Rosa roxburghii fruit;The high temperature treatment at 42 ? decreased the expression of GGP gene which could significantly reduce the AsA content of the fruit.However,the low temperature treatment at 4? played an opposite role.The Abscisic Acid(ABA)and MeJA treatments inhibited the activity of the GGP gene promoter deletions,and the GGP gene expression had a tendency to decrease.However,ABA treatment increased AsA content,and MeJA treatment decreased AsA content in Rosa roxburghii fruit.