Establishment Of VIGS System In Rosa Roxburghii Tratt And Effect Of RrGGP Silencing On Fruit AsA Biosynthesis

Rosa roxburghii Tratt.is widely concerned because its fruit is rich in vitamin C and other nutrients and active substances which have important medical and health care effects on human body.In recent years,the molecular biology and genetic engineering research of Rosa roxburghii Tratt,an important economic trait of fruit trees,has been gradually deepened,and more and more functional genes have been identified.However,the functional verification of these genes can not be achieved because the efficient regeneration system for genetic transformation of Rosa roxburghii Tratt has not been established.Virus induced gene silencing（VIGS）technology is to insert a target gene fragment into the virus vector.After infecting plant tissue,the expression of endogenous target gene homologous to the inserted fragment is inhibited and the corresponding character loss is shown,so as to quickly verify the function of target gene.Therefore,the establishment of VIGS technology system of Rosa roxburghii Tratt has an urgent need to explain and verify the function of key genes in key biological processes.In this study,the phytoene desaturase（Rr PDS）gene of Rosa roxburghii Tratt was used as the reporter gene,we established a VIGS system based on Tobacco Rattle Virus（TRV）and optimized the infection system to establish a new technology platform for rapid and efficient research on the gene function of Rosa roxburghii Tratt.On this basis,the VIGS technology was used to silence the expression of GDP-L-galactose phosphorylase（RrGGP）,the key gene of As A synthesis in Rosa roxburghii Tratt,in order to further verify its role in the process of As A synthesis in Rosa roxburghii Tratt.The main research results are as follows:1.Construction of Rosa roxburghii VIGS virus vector based on Tobacco Rattle Virus（TRV）vector.According to the transcriptome and genomic gene sequences,we have successfully cloned a 321bp PDS gene fragment and a385bp GGP gene fragment from Rosa roxburghii;the two fragments were connected with TRV virus vector respectively,and the recombinant vectors TRV2-Rr PDS and TRV2-RrGGP were obtained.were obtained.2.Establishment of VIGS system of Rosa roxburghii Tratt seedlings.The constructed recombinant vector TRV2-Rr PDS was transferred into Agrobacterium tumefaciens GV3101,and the concentration of OD₆₀₀was adjusted to 1.0.The stage of cotyledon of Rosa roxburghii Tratt seedlings were infected by injection method.After 48 days of inoculation,the leaves of Rosa roxburghii Tratt seedlings showed photobleaching phenomenon;the system also be used on the leaves of cutting branches,but the photobleaching phenomenon was not obvious when inoculated on the leaves of Rosa roxburghii Tratt seedlings.3.Optimization of Rosa roxburghii VIGS system.The effects of inoculation method,bacterial concentration OD₆₀₀values and culture temperature on PDS gene silencing of Rosa roxburghii seedlings were compared.The results showed that the suitable inoculation method for VIGS system of Rosa roxburghii seedlings was injection infiltration method,the suitable concentration of soaking solution was 1-1.5,and the suitable culture temperature was 25℃.4.RrGGP gene silencing in Rosa roxburghii Tratt fruit.The constructed TRV2-RrGGP vector of Rosa roxburghii Tratt was transferred into Agrobacterfium tumefaciens GV3101,and the fruit of Rosa roxburghii Tratt was infected by injection method,.After 48 days of inoculation,the expression level of RrGGP and the content of the As A were detected by q RT-PCR and HPLC.After silencing of RrGGP,the results showed that the expression of RrGGP decreased by 42.4%and the content of As A decreased by 30.1%.The results showed that GGP gene was the key gene of As A synthesis pathway.After silencing of RrGGP,q RT-PCR was used to detect the expression level of gene involved in As A biosynthetic pathway and metabolic pathway.The results showed that the gene involved inbiosynthetic pathway and metabolic pathway were affected after silencing.