Transcriptome Analysis And Functional Gene Validation Of Anthocyanin Over-accumulation Trait In Chinese Cabbage Leaves

Purple Chinese cabbage(Brassica rapa L.ssp.pekinensis)germline was created by distant hybridization and introgression breeding,using purple mustard(B juncea)as donor parent and green heading Chinese cabbage as recipient parent.It exhibits a phenotype of dark purple in leaves from inner to outer.The leaf cell extract contain large amont of cyanidin-based anthocyanin.In this study,we used RNA-seq transcriptome data and a powerful pipeline to investigate the molecular mechanism of the anthocyanin over-accumulation phenotype.Quality filtered reads were first mapped to B.rapa reference genome sequence,and lots of anthocyanin biosynthesis structural genes and a bHLH(Bra037887)regulatory gene was found significantly up-regulated among differentially expressed genes.This suggested that the anthocyanin pathway was thoroughly up-regulated.Then denovo assembled those unmapped reads,and obtained another set of DEGs.By doing sequence annotation,a R2R3-MYB coding transcript was identified as the key candidate gene,because of its high homology to a pivotal member PAP1 of anthocyanin transcriptional regulatory complex.Then molecular experiments were performed to validate those transcripts obtained from bioinformatics analysis,and gained several progressive results as follows.1.Chromosomal segment bearing c3563gli2 was not exist in green individuals of F2 population,but in purple individuals and materials with B genome.2.Based on a theory that some of the DEGs were caused by have-or-not of DNA fragments,through homologous sequence alignments and genome location and molecular markers,candidate gene along with the genome blocks U and E were duduced to transfer from B.juncea AABB genome to B.rapa AA genome.3.c3563gli2 and Bra037887 transcripts in parental purple B.juncea were significant highly expressed than in green B.juncea,with slight environmental effect and primary genetic effect.4.35S driven over-expression of c3563gli2 coding region in transformed Arabidopsis thaliana resulted in a purple phenotype of cotyledons.So,it can be concluded that this a exogenous candidate gene,which transferred among B.juncea and B.rapa genomes and simultaneously kept its high transcript abundance,constituted the underlying mechanism of anthocyanin over-accumulation trait in Chinese cabbage leaves.