The Light Response Of RrCDF3 In Rosa Roxburghii And Its Regulation On AsA Synthesis

The previous research of our group showed that GDP-L-galactose phosphotylase 2(Rr GGP2)is a key structural gene for the synthesis of high content of vitamin C(L-Ascorbic acid,As A)in Rosa roxburghii Tratt.A transcription factor of Dof gene family,Rr CDF3(cycling dof factor 3),was obtained by yeast one-hybrid(Y1H)screening using Rr GGP2 promoter.Bioinformatics predicted that it was related to light signals.Therefore,in this paper,R.roxburghii was used as the material to analyze the expression response of Rr CDF3 under bagging,compound light and photoperiod treatment,and to study its possible regulatory effect on Rr GGP2 through subcellular localization,dual-luciferase,Electro mobility shift assay(EMSA),transient overexpression and virus-induced gene silencing(VIGS),so as to provide more scientific basis for clarifying the transcriptional regulation mechanism of As A synthesis in R.roxburghii.The main results were as follows:1.Bioinformatics analysis and spatio-temporal expression characteristics of Dof gene family in R.roxburghii.19 genes encoding Dof proteins were identified from the whole genome of R.roxburghii.All of them contained a typical ZF-DOF domain.Gene structure analysis showed that six Rr Dofs genes had no introns,one had 4 introns,and the remaining members had 1-2 introns.The analysis of the physicochemical properties of the protein showed that most of the Rr Dofs proteins are alkaline and have a large molecular weight.Chromosome mapping analysis revealed that the Rr Dofs genes were unevenly distributed in seven chromosomes.The phylogenetic relationship and synteny analyses showed that Dof of R.roxburghii had a close evolutionary relationship with strawberry(Fragaria vesca)and Arabidopsis thaliana,and the whole Dofs of R.roxburghii and A.thaliana could be clustered into 9 different subclasses,respectively.The analysis of cis-acting elements in the promoter predicted that there were many cis acting elements in the promoter of Rr Dofs gene: light responsive elements were the most,followed by abscisic acid responsive elements and methyl jasmonate responsive elements,as well as anaerobic stress responsive elements,heat stress responsive elements,and low temperature stress responsive elements.RNA-seq and q RT-PCR analysis showed that Rr Dofs gene had spatio-temporal expression specificity in R.roxburghii,and correlation analysis showed that only Rr CDF3 was positively correlated with Rr GGP2 and As A content.2.Expression response of Rr CDF3 to different light treatments.We investigated the expression response of Rr CDF3 under different composite light qualities(white light,W;red blue light,RB;red blue white,RBW;red blue green,RBG)using 70 day after anthesis(DAA)fruits with branches,and the results showed that the expression of Rr CDF3 was regulated by both light quality and light/dark cycle.Overall,RB inhibited the expression of Rr CDF3,while adding white light to RB(RBW)can alleviate the inhibition of its expression by RB treatment.The expression of Rr CDF3 has a circadian rhythm,and its expression will resume after dark treatment.In addition,analysis of As A content after 6 days of treatment showed that incomplete light treatment was not conducive to the synthesis of As A.The different color fruit bags for bagging treatment also found that the expression of Rr CDF3 is influenced by the combined effects of light intensity and different transmission light qualities(wavelengths).Moreover,except for the consistent expression of Rr CDF3 and Rr GGP2 under natural light(CK)and complete shading(dark,D)treatments,there was no correlation under other color bagging treatments.These indicated that Rr GGP2 may not sense light quality signals through Rr CDF3.To gain a deeper understanding of the expression response of Rr CDF3 to photoperiod,we conducted three different photoperiod treatments on R.roxburghii seedlings(light/dark 16/8 h,12/12 h,8/16 h).By detecting the expression changes of these two genes and As A content in the leaves,it was found that Rr CDF3 significantly responded to light/dark duration.Under daily neutral conditions(12/12 h),the transcription levels of Rr CDF3 and Rr GGP2 were higher,and As A content accumulated more in the leaves.3.Direct Interaction Effect of Rr CDF3 and Rr GGP2 Promoters in Rosa roxburghii.Subcellular localization results showed that Rr CDF3 was located in the nucleus,which was consistent with the characteristics of transcription factors.Electrophoretic Mobility Shift Assay(EMSA)and dual-luciferase assay showed that Rr CDF3 could directly bind to the Rr GGP2 promoter and positively regulate the expression of Rr GGP2.Transient overexpression and VIGS silencing of Rr CDF3 on R.roxburghii fruit confirmed that Rr CDF3 can promote As A synthesis by positively regulating the expression of Rr GGP2,Transient overexpression of Rr CDF3 in strawberry fruits and tobacco leaves also had the same effect.