The Cloning And Functional Analysis Of The MYB1 And MYB10 Transcription Factors In Ribes L.

Currant belongs to the genus Ribes L.,family Saxifragaceae,its fruit products taste good and rich in nutrients.According to the agronomic traits,currents were divided into black currant(Ribes nigrum L.),red currant(Ribes rubrum L.)and white currant(Ribes albrum L.).Fruits colors were purple black,red and white.The fruit of black currant and red currant is rich in anthocyanins,which are a kind of natural water soluble flavonoid compounds.These kinds of flavonoid compounds are thought to fulfill important functions in scavenging free radicals,preventing cardiovascular diseases,neurological diseases,cancer and so on.Structural genes and regulatory genes are the two main categories that affect anthocyanin biosynthesis.To date,the R2R3-MYB transcription factor,bHLH-like transcription factor,and the MYB-bHLH-WD40(MBW)transcription complex that activates structural gene transcription in the anthocyanin pathway have been extensively studied.Many recent studies have shown that anthocyanin biosynthesis is controlled by transcriptional,post-transcriptional,and post-translational mechanisms.The R2R3-MYB protein in MBW complexes often plays a key role in anthocyanin biosynthesis and accumulation.To explain the molecular mechanism of differences in currant fruit coloration,three kinds of color of currant fruits were used as test materials.The RACE method was used to clone the full length sequence of the gene MYB1 related to anthocyanin synthesis of currant,and the gene characteristics and phylogenetic analysis of the currant MYB1 gene were analyzed.Real-time quantitative PCR was used to analyze the expression patterns of MYB1 and MYB10 genes in five different growth and development stages of currant fruit.The expression vectors of KYB1 and MYB10 genes were constructed respectively,and Arabidopsis thaliana was transfected by genetic transformation mediated by Agrobacterium,and positive transformants were successfully obtained and their phenotypes were observed.The results obtained are as follows:1.By the method of RACE,three full-length cDNA sequences of were anthocyanin biosynthesis-associated genes successfully cloned in the three kinds currant fruits.RnMYB1 and RrMYB1 were 985 bp and 975 bp in cDNA length encoding 303 amino acids.RaMYB1 was 917 bp in cDNA length encoding 130 amino acids.Phylogenetic analysis indicated that the amino acid sequence of RaMYB1 was quite different from other species,it was impossible to form an evolutionary tree.RnMYB1 and RrMYB1 were not similar to other species,belonging to one genus.2.The results of real-time quantitative PCR analysis showed that the expression of MYB1 and MYB10 in fruits was basically higher in black and red currant,and much higher than white currant.In black currant and red currant,the expression levels of RnMYB1,RrMYB1?RnMYB10 and RrMYB10 genes both increased first and then decreased,that is,75%reached the maximum before reaching maturity,and then the fruit was completely mature and decreased slightly.In white currant,the expression levels of RaMYBl and RaMYB10 were extremely low and almost undetectable,MYB1 and MYB10 genes play an important role in the coloring process of currant fruits.3.The Gateway technique was used to construct 6 gene overexpression vectors of MYB1 and MYB10 in three kinds of color of currant fruits respectively.After PCR detection and identification,the constructed recombinant plasmid expression vector was transferred into Agrobacterium LBA4404 by three-parent hybridization method.4.The Arabidopsis thaliana was infected by dipping,and transgenic plants transformed into RnMYB10,RrMYB10 and RaMYB10 were successfully obtained.The phenotypic observations on the transgenic lines showed that the RnMYB10 and RrMYB10 transgenic arabidopsis showed a clear purple petiole and leaves compared to the control arabidopsis.There was no obvious color change in the RaMYB10 transgenic arabidopsis.The expression-ofstructural genes related to anthocyanin biosynthesis,CHS,CHI,F3H,ANS,UFGT,and DFR,were all up-regulated,indicating that RnMYB10 and RrMYB10 genes had anthocyanin effects synthesis has a promoting effect.No significant changes in Arabidopsis thaliana transformed to RaMYB10.