Expression Of RrGGP Of Rosa Roxburghii Tratt In The Arabidopsis Thaliana And Its Effect

vitamin C also known as L-Ascorbic acid?AsA?or ascorbate,is one of the most important component of fruit quality.Previous work of the project team has proved that L-galactose pathway is the main pathway for AsA synthesis in Rosa roxburghii Tratt,which an important role of this gene in regulating AsA biosynthesis in the fruits.In order to further study the expression effect of the gene?and its promoter?,we constructed 35S::GUS,Pggp::GUS and 35S::RrGGP,Pggp::RrGGP expression vectors,and transformed them into Arabidopsis thaliana by inflorescence infiltration method and obtained transgenic plants.On the one hand,GUS was used as a reporter gene to analyze the expression of RrGGP promoter?Pggp?in transgenic plants,and on the other hand,to verify the functional effect of excessive expression of RrGGP in Arabidopsis thaliana on AsA synthesis and its response to some environmental factors and exogenous hormones.The main results were as follows:1.Vector construction,genetic transformation and screening of transgenic plants.The GUS and RrGGP gene was inserted into multiple cloning sites of the vector pFGC5941for obtaining the plant expression vector 35S::GUS and 35S::RrGGP.Based on that,replace35S sequence with Pggp promoter sequence for obtaining the plant expression vector Pggp::GUS and Pggp::RrGGP.The vector was transf-erred into Arabidopsis genome by the combined methods of Agrobacterium-mediated and pollen tube pathway.After the selection of basta and PCR detection,transgenic Arabidopsis plants of 35S::GUS?17?,Pggp::GUS?21?,35S::RrGGP?31?and Pggp::RrGGP?24?T₀ generation were successfully obtained.The positive transgenic Arabidopsis thaliana plants were screened by herbicide basta for 2generations and detected by PCR.2.Promoter activity of RrGGP promoter in transgenic Arabidopsis thaliana.The roots and leaves of 35S::GUS and Pggp::GUS transgenic lines were stained with GUS by histochemical staining?compared with non-transgenic wild type?WT?lines?.The results showed that GUS gene was strongly expressed in these two organs.However,the promoter activity of Pggp promoter was slightly weaker than that of 35S,the quantitative analysis of GUS activity confirmed the result of GUS protein staining,suggested promoter Pggp can initiate gene expression in transgenic Arabidopsis thaliana plants.The promoter activities of Pggp were not significantly induced when the transformed Arabidopsis thaliana exposing to MeJA.While the promoter activities of Pggp were significantly induced when treated by ABA,heat or drought.These results suggest that environmental conditions can regulate the expression level of corresponding genes by regulating the promoter activity of RrGGP.3.Expression of RrGGP in transgenic Arabidopsis thaliana.The transcription level of RrGGP gene and its effect on AsA content in 35S::RrGGP and Pggp::RrGGP transgenic lines were detected by qRT-PCR.The results showed that the RrGGP genes controlled by both promoters had up-regulation effect on expression compared with WT,and the content of AsA in transgenic plants increased by 1.54 times and 1.78 times compared with WT,respectively.Compared with untreated transgenic plants,the transcription level of RrGGP gene in Pggp::RrGGP transgenic plants under high temperature?40??and drought stress and ABA treatment was significantly increased,accompanied by a significant increase in AsA content,while the transcription level of RrGGP gene and plant was decreased by 100mmol L^-11 exogenous MeJA treatment.The content of AsA.These findings are of great significance to reveal the molecular mechanism of regulation of AsA synthesis in Rosa roxburghii by regulating the promoter activity of RrGGP promoter and the transcription level of RrGGP gene under environmental conditions.