Exploring Regulatory Genes Of Red Walnut(Juglans Regia L.) Anthocyanin Biosynthesis Based On Transcriptome Analysis

In order to reveal the mechanism of the red pigment in red walnut(Juglans regia L.)leaf and peel a green cultivar green walnut and a red cultivar red walnut were used in this study.The composition and the content of anthocyanin were analyzed.Transcriptome analyses was used to estimate of structural genes and transcription factors genes involved to anthocyanin biosynthesis at different developmental stages in the leaf and peel of red and green walnut.Furthermore,the selected genes and transcription factors were conducted via q RT-PCR.The main results were as follows: 1.A method of ultra performance liquid chromatography with photodiode array detection coupled with electrospray ionization tandem mass spectrometry(UPLC-PDA-MS/MS)was used for the detection of anthocyanins in red walnut.The composition and the content of anthocyanin in leaf and peel in red walnut and green walnut were analyzed by this method.The result showed that eight anthocyanins were detected from the red cultivar leaf of red walnut.They are delphinidin-3-O-galactoside,delphinidin-3-O-glucoside,cyanidin-3-O-galactoside,delphinidin-3-O-arabinoside,cyanidin-3-O-glucosid,cyanidin-3-O-arabinoside,delphinidin-3-O-xyloside,cyanidin-3-O-xyloside.The composition and content of anthocyanins in leaf are different in the different periods.delphinidin-3-O-galactoside and cyanidin-3-O-galactoside are the two main types of anthocyanin in the anagen of red walnut leaf.The content of 8 anthocyanins had been declining in the growing process,which was consistent with the actual growth process phenomenon of red walnut leaves.Only delphinidin-3-O-glucoside was detected in the growing and mature stage of red walnut peel,this kind of anthocyanin showed an upward trend in the growing whichwas consistent with the actual growth process phenomenon of red walnut peel.What's more,results showed that although the less content of delphinidin-3-O-glucoside in the peel of red walnut,maybe it's the main content which can regulate the color of red walnut peel.This is an obvious differences in the peel of red and green walnut.None of anthocyanin was detected in the leaf and peel of green walnut.2.Normalized c DNA libraries from red cultivar red walnut and green cultivar green walnut leaf and peel were sequenced using the Illumina Hiseq 2500 sequencing platform and assembly.544,888,472 raw reads were annotation into 30,648 genes in the leaf of walnut.Sequence homology revealed that 19,520 genes were assigned to GO term.17,701 genes were assigned to 26 COG.And 11,074 genes were assigned to 242 KEGG pathways.423,238,369 raw reads were annotation into 29,370 genes in the peel of walnut.Sequence homology revealed that 19,080 genes were assigned to GO term.17,389 genes were assigned to 26 COG.And 7,477 genes were assigned to 242 KEGG pathways.3.To investigate the gene expression patterns in the leaf and peel of red and green walnut during the growing process,eight DGE libraries in the leaf of red and green walnut and six libraries in the peel of red and green walnut were constructed and sequenced using Illumina Hiseq 2500 deep sequencing technology.It finds that the expression of genes encoding PAL,4CL,F3 H,F3'5'H,DFR,ANS and UFGT in red walnut showed significantly up-regulated during the growing process in the leaf of red and green walnut.The expression of genes encoding 4CL,CHS,CHI,F3 H,F3'5'H,DFR,ANS and UFGT in red walnut showed significantly up-regulated during the growing process.The gene PAL Jrgene11958 in the peel and the gene CHS Jrgene4994 in the leaf showed significantly down-regulated in the red and green walnut.Furthermore,we identified that 3 candidate MYB genes,2 candidate b HLH genes and 2 candidate WD40 gene among the DEGs which may regulate anthocyanin biosynthesis in the leaf of red and green walnut.What's more,we identified that 3 candidate MYB genes,2 candidate b HLH genes and 3 candidate WD40 gene among the DEGs that may regulate anthocyanin biosynthesis in the peel of red and green walnut.We confirmed fifteen genes in the leaf and fifteen genes in the peel including enzymes and transcription factors using q RT-PCR.