Cloning And Expression Analysis Of R₂R₃-MYB Genes Related To Anthocyanin Biosynthesis In Rosa Rugosa

Rosa rugosa is a deciduous shrub of genus Rosa in the Rosacea family with highly ornamental value,and it plays an important role in landscaping.The flower color of R.rugoa is very single,most is red,pink,and white,other colors are rarely seen,which has seriously limited its application in landscaping.Anthocyanin is one of water-soluble natural pigments widely existing in flowers,fruits,stems,leaves and seeds in natural plants,and plays an important role in the color of R.rugosa.Anthocyanin is synthesized through the synthetic pathway of flavonoids in phenylpropane pathway,and it is usually catalysed with a series of synthetase and transport proteins.In higher plants,R₂R₃-MYB,bHLH,WD40 are three important TFs of regulating anthocyanin synthesis.Furthermore,As the most widely used transcription factor in anthocyanin synthesis,R₂R₃-MYB protein can activate one or more structural genes expression,thereby promoting anthocyanin synthesis and making plants red or purple.At present,there are few studies on the molecular regulation mechanism of anthocyanin synthesis in R.rugosa.Therefore,cloning the MYB TFs from R.rugosa that related to anthocyanin synthesis is important for understanding the regulation mechanism of anthocyanin accumulation and changing the colors.Using R.rugosa‘Zi zhi’,R.rugosa‘Bai zizhi’,R.rugosa‘Fen zizhi’as the materials,we cloned three MYB TFs,RrMYB6,RrMYB10,RrMYB113,and obtained the total length of cDNA of them by RT-PCR and RACE.The method of bioinformatics is used to analyze and identify the sequence structure and physical of the three MYB TFs.Using real-time quantitative PCR（qRT-PCR）to analyze the expression of the three MYB TFs in different tissues and different varieties.The main results are as follows:1.Three R₂R₃-MYB TFs were cloned from the petals of R.rugosa‘Zi zhi’in bud period:RrMYB6,GeneBank accession Nos:MG745778,has an open reading frame（ORF）with length of 921bp,and encoding 306 amino acids;RrMYB10,GeneBank accession Nos:MG745779,has an ORF of 699 bp,and encoding 232 amino acids;RrMYB113,GeneBank accession Nos:MG720012,encoding 216 amino acids with an ORF of 651bp.2.Bioinformatics analysis showed that all three MYB transcription factors contained R2,R3 domain and bHLH binding domain,which were typical R₂R₃-MYB transcription factors.In addition,RrMYB6 also had C1 and C2 suppressor motif domain and PCCEK（N1）motif,thus belonging to R₂R₃-MYB protein in Sg4 subfamily.What’s more,RrMYB10 and RrMYB113 proteins had（A/S/G）NDV and KPRPR（T/S）motifs and belonged to Sg6subfamily.The formulas of proteins encoded by RrMYB6,RrMYB10,RrMYB113 were C1491H2368N452O470S17,C1193H1887N347O353S8 and C1109H1747N333O329S9 respectively.The prediction results showed that there were mainly alpha-helix and Random coil in the secondary structures of the three MYB proteins.The results showed that all three MYB proteins were unstable hydrophilic proteins,and they also belonged to non-secreted proteins with a large number of phosphorylation sites and no transmembrane domain.3.The qRT-PCR results showed that three MYB genes all expressed in different tissues and varieties,but the expression patterns were significantly different.The results of the comprehensive experiment suggested that all three MYB genes were involved in the anthocyanin synthesis of R.rugosa.And RrMYB6 and RrMYB10 were negative regulatory factors,while RrMYB113 was a positive regulator.In addition,it is speculated that RrMYB6and RrMYB10 were also involved in regulating the growth and development of R.rugose.4.ThreeMYBgeneexpressionvectorspCAMBIA1304-RrMYB113,pCAMBIA1304-RrMYB6 and pCAMBIA1304-RrMYB10 were successfully constructed and transformed into Agrobacterium GV3101.In future work,we will test whether the overexpression of these genes leads to anthocyanin accumulation in Arabidopsis thaliana and Nicotiana tabacum.