The Key Structural Genes Analysis Of Anthocyanin Biosynthesis In Red-fleshed Kiwifruit(Actinidia Arguta) Based On Transcriptome Sequencing

Flesh color is an important fruit characteristic,and is one of the main factors that will affect consumers whether purchase it or not.At present,there are more studys about chlorophyll metabolism on green-fleshed and carotenoid metabolism on yellow-fleshed kiwifruit.However,relatively less research about anthocyanin metabolism on red-fleshed kiwifruit.Furthermore,‘Hongyang' is often selected as materials during some related studys about anthocyanin metabolism,no all red-fleshed kiwifruit is involved.‘Tianyuanhong(TY)' is obtained through natural selective breeding.In addition to some advantages such as early maturity,glabrous pericarp and flesh,and the core,all of which turn red when it is ripe,TY is edible even without the course of afterripening.In this study,‘TY' was selected as the experimental material,with the green-fleshed cultivar ‘Yongfengyihao(YF)'as the control.Color parameters a,b at 8 stages after full bloom were measured with Konica Minolta portable color meter CR-400.High-performance liquid chromatography(HPLC)was also used for semi-quantitative analysis on these fruits at different stages so as to explore the difference of anthocyanin at different stages.RNA-seqencing and data-screening were conducted to find out structural genes that play a key role in anthocyanin biosynthesis,which would provide gene resources for the genetic improvement.As light is a crucial factor that can influence the fruit color,we conducted three bagging treatments on ‘TY' kiwifruit,which evaluated the effect of bagging on fruit pigmentation in order to provide evidence for the impacts of light on anthocyanin synthesis.The main results of this study were as follows:The a and b measured by CR400 at 8 stages after full bloom were applied to calculate the color ratio(h=a/b)and hue angle(h*=arc[tan(b/a)]),which fially showed that degrenning and reddening the fruit during its growth and development.Semi-quantitative analysis was conducted at 8 stages with standard samples of cyaniding-3-galactoside(Solarbio,Beijing).The results showed that anthocyanin content increased in the fruit of ‘TY' increased gradually from 90d(days after full bloom)and reached the peak at 120 d.The bagging treatment could dramatically decrease the hue angle of the epicarp of ‘TY',while it had less effect on sarcocarp and columella.The results showed that h value and hue angle had significant difference between different treatments,different parts and different stages.Bagging could inhibit the accumulation of anthocyanins in the epicarp,outer pericarp,inner pericarp(sarcocarp)and columella.The anthocyanin content in the epicarp and sarcocarp in the bagging-debagging treatment increased significantly after debagging,and reached the peak value at 110 d,when the anthocyanin content in the epicarp and sarcocarp of debagged fruit was significantly higher than that of non-bagged and bagged fruits,while the anthocyanin contedt in the columella was not.The difference in anthocyanin content in different treatments,different fruit parts and different stages reached significant levels.We performed RNA-seq to obtain 329,189 transcripts with a total length of 252,165,247 and N50 of 1,233 and N90 of 306.After assembly and splice of those initial transcripts,we finally obtained 202,742 unigenes with a total length of 122,182,370 and N50 of 873 and N90 of 255.Of these unigenes,72,508(35.76%)were annotated in seven databases(NR,NT,KO,Swiss Prot,PFAM,GO,KOG),and 572 unigenes were assigned to 13 secondary metabolic pathways,of which 104 unigenes were involved in flavonoid and anthocyanin biosynthesis.Through the enrichment analysis of differential expression of differentially expressed genes in different samples and the parameter log2fold-change and p-adjusted,12 DEGs(differentially expressed genes)were finally selected.The gene id were: c130230_g1,c126583_g2,c123355_g1,c114683_g2,c110715_g4,c120031_g4,c117486_g2,c118500_g1,c128188_g2,c120091_g5,c130006_g1,c108941_g2;gene name were: PAL,C4 H,4CL,CHI,F3 H,LDOX,FLS,F3'H2,HCT,C3'H,CCOMT,F3 GT.The 12 selected DEGs were conducted to analysis on the expression rule at the eight stages using q RT-PCR(quantitative real-time polymerase chain reaction).The results showed that the gene expression level of F3 H and LDOX(genes encoding flavanone-3-hydroxylase and leucoanthocyanidin dioxygenase,respectively)were highest on 120 d.This was the stage at which the kiwifruit flesh evidently turned red,suggesting that these two genes may play an important role in the red color change phenomenon.The results of cluster analysis showed that LDOX was clusted into one class,which was obviously separated from other genes.Thus we inferred that LDOX might be the key structural genes during the process of anthocyanin biosynthesis in ‘TY' kiwifruit.