Expression Pattern And Functional Analysis Of RrTTG1 In Rosa Roxburghii Tratt

Rosa roxburghii Tratt belongs to the Rosaceae,and its fruit resembles a pear and has dense small thorns on the surface.The fruit of R.roxburghii has a unique taste and rich nutrition,especially riches in antioxidants such as vitamin C and flavonoids.R.roxburghii Tratt is widely grown in Guizhou Province.Fruit thorns,antioxidants,and flowering are essential quality characteristics related to fruit market value,but it remains obscure about the genes associated with these characteristics in R.roxburghii.In Arabidopsis thaliana,TRANSPARENT TESTA GLABRA1（TTG1）gene regulates multiple traits related to plant adaptation to environmental changes,including the initiation of trichomes and root hair,anthocyanin accumulation,seed coat color formation,and flowering.Therefore,it is of great significance to study the function of the RrTTG1 in these processes in R.roxburghii.In this study,the cDNA of RrTTG1was obtained by cloning.Then,its expression pattern and subcell location were analyzed.Both genetic transformation and transient transformation were performed to study the biological function of RrTTG1 further.Finally,a Y2H（yeast two hybrid）and Bi FC（bimolecular fluorescence complementation）were used to analyze the interaction of RrTTG1 with b HLH proteins.The main results obtained are as follows:1.The length of RrTTG1 CDS was 1041 bp,and RrTTG1 encoded a hydrophilic protein containing 346 amino acids.The molecular size of RrTTG1was 38.63 k Da,and its PI was 4.95.The domain analysis of RrTTG1 showed that it contains 4 WD40 repeat domains and belongs to the WD40 protein family.The phylogenetic tree indicated that RrTTG1 has high homology with TTG1 of Rosaceae,such as Rosa rugosa,Rosa chinensis,Fragaria x ananassa,Fragaria vesca,and Rubus idaeus,the homology is 99%,98%,96%,96%,and 94%respectively.The homology with Arabidopsis is 79%.2.The expression level of RrTTG1 in various vegetative organs was detected by q RT-PCR and in situ hybridization.The results showed that the expression level of RrTTG1 in leaves was the highest and followed by that in flower buds.The results of in situ hybridization were consistent with the q RT-PCR results.Meanwhile,it has found that the m RNA of RrTTG1 was enriched in a vascular bundle,suggesting that its m RNA may transport through the vascular bundle,thereby participating in other tissues or organs’development.Next,the expression patterns of RrTTG1 during fruit development were detected.The results of q RT-PCR showed that RrTTG1 maintained a high expression level during fruit development,especially in the early stage.In situ hybridization showed that the signal of RrTTG1 m RNA was enriched in bracteoles inside flower buds,which may be related to anthocyanin synthesis regulation.Meanwhile,RrTTG1 was also highly expressed in fruit spurs,which provided evidence for RrTTG1 to participate in fruit spurs development.3.We constructed a CaMV35S::RrTTG1:GFP vector,transformed it into Arabidopsis protoplasts with PEG4000,and used DAPI to label the nucleus and Dil to label the cell membrane.The green fluorescent signal of RrTTG1:GFP was only observed in the nucleus by LSCM,indicating that RrTTG1 was localized in the cell nucleus and was a transcription factor.4.We constructed an overexpression vector RrTTG1-p BI121.After transforming the inflorescence of Arabidopsis thaliana,eight overexpression transgenic plants,named OXRrTTG1-1～OXRrTTG1-8,were obtained.Three T₂overexpression plants（OXRrTTG1-6,OXRrTTG1-7,and OXRrTTG1-8）were selected for further phenotypic observation.Up-regulated the expression of RrTTG1 would cause multi-phenotypes,including increased trichrome,less root hair,darken seed coat,early flowering,and increased anthocyanin.Using HPLC to detect the content of anthocyanin in hypocotyls of 5d-seedlings,it had confirmed that its content was improved obviously.5.We constructed an RNAi vector RrTTG1-pDS1301.The overexpression vector and RNAi vector were transferred into Agrobacterium GV3101,respectively.Then the transformed Agrobacterium was injected into the fruits of R.roxburghii by transient transformation method to obtain the OXRrTTG1 and RrTTG1-RNAi fruits,respectively.q RT-PCR results showed that the expression of RrTTG1 increased in 30 DAI（days after injection）OXRrTTG1 fruit,and decreased in 30 DAI RrTTG1-RNAi fruit.Meanwhile,the number of fruit thorns increased when the expression of RrTTG1 was up-regulated,while reduced when its expression was knock-down.6.Y2H and BiFC were used to detect the interaction of RrTTG1 with b HLH proteins At GL3,At EGL3,and their homologs（RrGL3 and RrEGL3）.The results revealed that RrTTG1 could interact with At GL3 and At EGL3,indicating that the function of TTG1 was conserved during the evolutionary process.Interestingly,we found that RrTTG1 can only interact with RrEGL3 but not with RrGL3,suggesting that RrEGL3 was functionally conserved during evolution,while RrGL3 might have differentiation.