| Anthocyanins are branch products of flavonoids synthesis pathway,and light is the most important environmental factor that affects the anthocyanin biosynthesis.Eggplant fruit is rich in nutritions and anthocyanins.The peel of eggplant coloring poor under the weak light condition in actual production,and caused quality declined.However,due to the limitations of conventional sequencing technology,research of the mechanism of eggplant fruit pigmenting by light is rare.Thus,light-sensitive eggplant cultivar ‘Lanshan Hexian' was used as research material,analysis light-induced response mechanism through bagging treatment,RNA-seq and iTRAQ technology from mRNA and protein level in eggplant.The main research results are as follow:1)Identified two kinds of anthocyanin components by ultra performance liquid chromatography-tandem mass spectrometry(UPLC-MS/MS)in ‘Lanshan Hexian':delphinidin-3-[4-(cis-p-coumaroyl)-rhamnosyl-glucopyranoside]-5-glucopyranoside,delphinidin-3-[4-(trans-p-coumaroyl)-rhamnosyl-glucopyranoside]-5-glucopyranoside.The relation curve of eggplant peel anthocyanin content and time displayed “S” type,and the curve inflection was 5.191 d.Thus,the time points of sampling for RNA-seq and iTRAQ were determing(0 d,5 d,12d).2)It has found through RNA-seq that 784 differentially expressed genes(DEGs)were detected between 5 d and 0 d libraries.According to the GO functional enrichment analysis,784 DEGs were divided into 30 functional categories.According to the KEGG Pathway functional enrichment analysis,43 significantly enriched pathways were obtained,including flavonoids biosynthesis pathway.516 DEGs were detected between 12 d and 5 d libraries.According to the GO functional enrichment analysis,516 DEGs were divided into 27 functional categories.According to the KEGG Pathway functional enrichment analysis,29 significantly enriched pathways were obtained,also including flavonoids biosynthesis pathway.Further analysis found that the expression pattern of the genes which annotated to the anthocyanin biosynthesis structure genes(CHS,CHI,F3 H,F3'5'H,DFR,ANS)and regulatory gene(MYB1)was up-regulated from 0 d to 5 d,and then down--regulated from 5 d to12 d.It explained the possible mechanism of light regulating eggplant anthocyaninsynthesis in mRNA level.3)It has found through iTRAQ that 293 differentially expressed proteins(DEPs)were detected between 5 d and 0 d libraries.According to the GO functional enrichment analysis,293 DEPs were divided into 37 functional categories.According to the KEGG Pathway functional enrichment analysis,23 significantly enriched pathways were obtained.Unlike RNA-seq data,DEPs did not significant enriched on flavonoids biosynthesis pathway.228 DEPs were detected between 12 d and 5 d libraries,and among them.According to the GO functional enrichment analysis,228 DEPs were divided into 34 functional categories.According to the KEGG Pathway functional enrichment analysis,11 significantly enriched pathways were obtained,and also DEPs did not significant enriched on flavonoids biosynthesis pathway.Further analysis found that only CHI and F3 H were identified in DEPs.4)According to the correlation analysis of transcriptom and proteome data,52(5d vs 0 d)and 39(12 d vs 5 d)associated DEPs were discovered,respectively.In 5 d vs 0 d,the proteins of CHS,F3 H,F3'5'H,DFR and ANS were not differential expressing,and their genes were differential expressing;Both the proteins and genes of CHI were differential expressing.In 12 d vs 5 d,the proteins of CHS,F3'5'H,DFR and ANS were not differential expressing,and their genes were differential expressing;both the proteins and genes of CHI and F3 H were differential expressing.The result reflected that most of the expression patterns of anthocyanin biosynthesis genes' proteins did not agree with mRNA.5)In order to study the changes of the mRNA expression level in short-time after photoinduction,RNA-seq was adopted to analyse the transcriptome information in 0 h,0.5 h,4 h and 8 h after exposed to light.843 DEGs were detected between 0.5 h and 0h libraries.According to the GO functional enrichment analysis,843 DEGs were divided into 31 functional categories.According to the KEGG Pathway functional enrichment analysis,30 significantly enriched pathways were obtained.865 DEGs were detected between 4 h and 0.5 h libraries.According to the GO functional enrichment analysis,865 DEGs were divided into 31 functional categories.According to the KEGG Pathway functional enrichment analysis,28 significantly enriched pathways were obtained.223 DEGs were detected between 8 h and 4 h libraries.According to the GO functional enrichment analysis,223 DEGs were divided into 27 functional categories.According to the KEGG Pathway functional enrichment analysis,18 significantly enriched pathways were obtained.Further analysis found that the expression pattern of the genes which annotated to the anthocyaninbiosynthesis structure genes(CHS,CHI,F3 H,F3'5'H,DFR,ANS),regulatory gene(MYB1)and positive light signal regulatory factor(HY5)was no significant change or slightly up-regulated in the expression level from 0 h to 0.5 h,and then sharply up-regulated from 0.5 h to 4 h,and no significant change or slightly down-regulated from 4 h to 8 h,reflecting obvious light-inducing.6)Full length of CHS,CHI and F3 H were cloned from eggplant.Together with the DFR cloned from laboratory in earlier stage and F3'5'H and ANS from NCBI,the exon-intron architecture of the six genes were analyzed firstly.qRT-PCR displayed that all the six genes were ubiquitously expressed in roots,stems,leaves,petals,peel and flesh.The highest expression levels of CHS,CHI,F3'5'H,DFR and ANS were observed in peels.In addition,the highest expression level of F3 H was detected in stems.Further analysis discovered that anthocyanin content showed significant positive correlation with the expression levels of CHS,CHI,F3'5'H,DFR and ANS through correlation analysis,except for F3 H.The expression of CHI,F3'5'H,DFR and ANS was barely detectable in bagged peels(Fig.4).The expression of CHS and F3 H in normal peels was 4.50-and 51.94-fold than bagged peels.All the six anthocyanin biosynthesis genes were up-regulated in cold stress experiment,and SmCHS increased most obviously.Subcellular localization suggested that the enzymes encoded by CHS,CHI,F3 H and DFR were all located in cytosol and nucleus.The overexpression Arabidopsis lines of CHS,CHI,F3 H and DFR displayed higher anthocyanin accumulation in stems and siliques.7)Two blue light receptor genes,CRY1 and CRY2,and negative/positive light signal regulatory factors COP1 and HY5 were isolated from eggplant.Together with MYB1,which has been proved to play an important role in activating anthocyanin biosynthesis in eggplant,CRY1 expressed at the highest level in leaves,CRY2 expressed at the highest level in roots and leaves,COP1 expressed at the highest level in petals,MYB1 expressed at the highest level in peel,while HY5 displayed no tissue-specific.The expression of CRY1,CRY2,HY5 and MYB1 are induced by light,and the expression of COP1 is inhibited by light.The transgenic seedlings ectopically expressing CRY1 and CRY2 partially recovered the hypocotyl length phenotype under blue light conditions,and CRY2 recovered the flowering time.The transgenic seedlings ectopically expressing COP1 and HY5 inhibited anthocyanin accumulation and recovered the cotyledon and hypocotyl length phenotype.The results indicated that the 4 genes have CRY1,CRY2,COP1 and HY5 functions.Yeast two-hybrid and bimolecular fluorescence complementation assays demonstrated that CRY1 andCRY2 interact with COP1 in a blue-light-dependent manner in yeast and plant.Also,COP1 interacts with HY5 and MYB1 in yeast and plant.The result of yeast one-hybrid assay revealed that both HY5 and MYB1 bind to the promoters of CHS and DFR.Thus,a new regulation pathway describing the possible mechanism underlying the light-controlled anthocyanin biosynthesis in eggplant has been proposed.The light receptors CRY1 and CRY2 perceive light signals,and interact with COP1.As a result,HY5 and MYB1 accumulate and directly bind to the promoters of CHS and DFR to activate their expression.Finally,anthocyanin biosynthesis leads to coloration in eggplant fruit.In darkness,CRY1 and CRY2 can't interact with COP1,then COP1 interacts with HY5 and MYB1 and mediates them degradation.Thus,downstream anthocyanin biosynthesis structure genes can't be activated,and no anthocyanin would be synthesized. |
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