Cloning And Expression Analysis Of MYB Transcription Factor In Blood Orange(Citrus Sinensis)

Anthocyanins belong to a kind of flavonoid with biological activity and are widely found in different plants.Anthocyanins have strong anti-oxidation activity and are responsible for the colors of most fruits,vegetables,flowers,and cereal grains.Blood orange(Citrus sinensis)is an important cultivar of sweet orange rich in anthocyanin.In this study,three varieties of blood orange were used to compare the fruit quality and anthocyanin content changes were found during fruit development.And the full-length coding sequence of MYB type transcription factor was cloned from juice vesicles in blood orange,bioinformatic analysis and gene profiling were performed.Meanwhile,prokaryotic expression of CsMYB was used to immunize animals to prepare polyclonal antibody.Then Western blot was performed to detect CsMYB differential expression among samples.Furthermore,the yeast two-hybrid technique was conducted to study the interactions between CsMYB and other regulators.The main results were obtained as follows:(1)Comparative analysis of fruit quality between different varietiesThe internal quality of fruit in three varieties was significantly different.The content of total anthocyanin and the soluble solids in 'Zaohong blood orange' was the highest.The titratable acid content of 'Wanhong blood orange' was the highest.The soluble sugar content of 'No.3 blood orange' was the highest.There was no significant difference in the external quality of fruit in three varieties.At different developmental stages of 'Zaohong blood orange',the anthocyanin content was significantly different.When the fruit was young(131 days after flowering),the content of anthocyanin was low in 0.48 mg/L.And at the fruit-coloring one stage(238 days after flowering),the anthocyanin content was 6.006 mg/L.While at the fruit delayed-harvest for two months stage(302 days after flowering),the anthocyanin content rose rapidly to 85.32 mg/L.(2)Cloning and bioinformatics analysis of CsMYB in the juice vesiclesThe MYB transcription factor was cloned by homologous cloning from the cDNA of juice vesicles.The full-length coding sequence of MYB was 789 bp,which encoded a protein with 262 amino acids,with R2 and R3 MYB conserved domains,so that it was designated by CsMYB(Gen Bank No.KT757348).The N-terminal of CsMYB contains a conserved motif [DE]Lx2[RK]x3Lx6Lx3R that interacts with the bHLH transcription factor.The C-terminal contains a conserved motif KPRPR[S/T] [F/L] that regulates anthocyanin synthesis.The phylogenetic tree performed using different homologous MYB-type proteins shows that CsMYB protein was clustered with litchi Lc MYB1 protein,and the amino acid identity between the two proteins was 56.1%.CsMYB genome sequence and promoter sequence were cloned from the DNA of blood orange leaves.The CsMYB genomic sequence is 1681 bp with three exons and two introns.And the promoter sequence(750 bp)consists of 5' UTR and LTR,including multiple low-temperature response elements,light response elements,and stress response elements.(3)Differential expression profiling of important genes in blood orangeThe level of CsMYB expression was exited significant differences in three varieties of blood orange.The expression of CsMYB was the highest in BO9 with high anthocyanin content.The Pearson's correlation coefficients of the anthocyanin content in juice and the relative expression of CsMYB,CsbHLH,CHS in juice vesicles were 0.805(p?0.01),0.762(p?0.05),0.772(p?0.05),respectively.Comparison of CsMYB transcription in different tissues of blood orange demonstrated that the higher transcription level was found in the juice vesicles.Besides,CsbHLH had the same expression pattern.While Cs WD40 was expressed high in all tissues.The gene expression of CsMYB and CsbHLH were significantly increased during fruit development,and the expression changes of the genes which are involved in the anthocyanin biosynthesis pathway,like CHS(Chalcone synthase),ANS(Anthocyanidin synthase),DFR(Dihydroflavonol-4-reductase),and LDOX(Leucoanthocyanidin dioxygenase)were in the same tendency with former.In addition,the expression of GST(Glutathione S-transferase)which involved in vacuolar anthocyanin transporter was gradually increased.In flavedo color mutational chimera,the expression levels of CsMYB and CsbHLH in red flavedo were higher than in yellow flavedo,and CHS and DFR co-expressed with the regulatory genes.However,the anthocyanin content in the juice vesicles of the red flavedo and yellow flavedo was no difference,the expression trend of CsMYB,CsbHLH,DFR,CHS,and other genes was same as the change of anthocyanin content.(4)Preparation of polyclonal antibody and Western blot detection on CsMYBThe prokaryotic expression vector p ET28-CsMYB was developed and transferred into Escherichia coli BL21 to obtain prokaryotic expression engineering strain.CsMYB recombinant protein was extracted and used to immunize animals to obtain polyclonal antibody.Western blot detection of CsMYB recombinant protein using the prepared antibody,the antibody specifically binds the antigen at 36 k D with a single band.What's more,Western blot result showed that the CsMYB protein was only expressed in the red-fleshed fruit.(5)Interactions between MYB,bHLH,WD40 transcription factors in blood orangeThe yeast two-hybrid assay indicated that CsMYB protein could not interact with CsbHLH protein directly,while Cs WD40 protein interacted with CsMYB protein and CsbHLH protein respectively.Taken together,CsMYB might be involved in the anthocyanin regulation of blood orange.This study could provide an insight about of fruit coloring mechanism and genetic breeding of blood orange.