Identification And Functional Analysis Of R2R3-MYB Regulators In The Anthocyanin Biosynthetic Pathway From Prunus Mume

Flower color is an important economic trait of Prunus mume,a famous ornamental plant widely cultivated in China.Flower color breeding is one of the important goals of P.mume breeding.However,very little was known about systematic studies on the formation mechanism of flower color of P.mume,and the metabolic pathway and regulation mechanism of flower color were not clear.Anthocyanins,acting as major pigments of flower coloration,are biosynthesized via transcriptional regulation of transcription factors(TFs).Many R2R3-MYB TFs have been identified to regulate anthocyanin biosynthetic pathways.However,very little is known about the role of R2R3-MYB TFs regulating anthocyanin biosynthesis in P.mume.The publication of P.mume genome lays the foundation for studying the molecular mechanism of flower color formation.In this paper,the composition of anthocyanins and their effect on flower color were studied,and the anthocyanin synthesis pathway was clarified;According to the genome data of P.mume,the MYB gene family in P.mume was identified and the R2R3-MYB gene related to anthocyanin regulation was predicted and screened by bioinformatics methods.Then these genes were isolated from P mume by RT-PCR method and transferred into tobacco by constructing expression vectors to elucidate their functions.Subsequently,the effects of over-expression of these genes on the phenotype and endogenous anthocyanin biosynthesis genes of transgenic tobacco were analyzed.The main results of this paper are as follows:(1)The flower colors of 41 P.mume varieties were divided into four groups:white,pale pink,pink and purple.No anthocyanin component was detected in the white series,and 5 anthocyanin components were detected in other color series.The 4 major components were identified,which accounted for more than 99%of total anthocyanin content.These components included cyanid in-3-O-glucoside(Cy3G),cyanidin-3-O-rhamnosyl-glucoside(Cy3GRh),Peonidin-3-O-glucoside(Pn3G)and Peonidin-3-O-rhamnosyl-glucoside(Pn3GRh).Therefore,it was concluded that the main anthocyanins metabolic pathway of P.mume was the branch of the cyanidin.The average contents of Cy3GRh,Pn3GRh and Cy3G accounted for 93.5%of the total anthocyanin content,as the main anthocyanin component of P.mume,has an important effect on the phenotype of its flower color.(2)96 R2R3-MYB genes were identified from the P.mune genome and divided into 35 subfamilies via phylogenetic analysis with Arabidopsis thaliana.The results of bioinformatics analysis of 96 genes as follows:each sequence of 96 genes contains a conserved MYB domain;79.2%genes contain 2 introns in their sequence structure;the 87 genes were not evenly distributed on the 8 chromosomes of P.mume,and the number of genes on chromosome 4,2 and 7 was the highest;segment repeats and tandem repeats played important roles in the expansion of PmMYB family;most of the R2R3-MYB genes were expressed in different organs,which may be involved in the development of different organs of P.mume.It was speculated that there may be four R2R3-MYB genes involved in anthocyanin synthesis.(3)The four CDS sequence with total length of 741,579,729,and 582 bp were obtained by RT-PCR method from P.mume 'Hongxu Zhusha',respectively.Multiple sequence alignment and phylogenetic analysis were performed between the four CDS sequences with R2R3-MYB genes involved in anthocyanin regulated from other species.The results showed that the four CDS sequences had high homology with the R2R3-MYB genes regulating anthocyanin synthesis from apple,pear,sweet cherry and peach in Rosaceae.Therefore,it was speculated that they may have similar biological functions.(4)The function of PmMYB1 and PmMYBal was identified by over-expression in tobacco,which significantly promoted the accumulation of anthocyanin in transgenic tobacco.The flower colors of overexpressing PmMYB1 transgenic plants were significantly deepened,and the anthocyanin contents in the corolla of transgenic plants were significantly higher than that of the control(P<0.05),their average value was 6.2 times that of the control.The flower color and fruit color of overexpressing PmMYBal tobacco plants were significantly enhanced,and the anthocyanin content in the corolla and pericarp of transgenic plants were significantly higher than those of the control(P<0.05).The mean values were 5.2 and 5.4 times that of the control,respectively.Gene expression analysis showed that most endogenous anthocyanin synthesis pathway genes were significantly up-regulated in transgenic tobacco flowers.In addition,both PmMYB1 and PmMYBal genes were stably expressed and obvious phenotypic changes were also maintained in transgenic tobacco progeny.These results indicated that the two genes played an important role in the regulation of anthocyanin synthesis.