Waxy Related Differential Proteomic Study Of 'Ganqi 3' Navel Orange During Fruit Development And Cold Storage By ITRAQ Technology

‘Ganqi 3' navel orange is a bud mutant cultivar from ‘Newhall' navel orange.The major phenotype trait of ‘Ganqi 3' is the unusual glossy fruit peels.To explore the mechanism of wax content and cold storage(4?)ability differences between wax mutant‘Ganqi 3' and wild type ‘Newhall' at the protein level,iTRAQ technology were used to study the proteomic difference between the ‘Ganqi 3' and ‘Newhall' navel orange fruit peels sampled during fruit development and cold storage.In addition,the combined analysis of proteomic and previous transcriptome data were performed to screened the wax related differentially expressed pathways and differentially expressed proteins(DEPs).Meanwhile,the expression of DEPs at mRNA level were examined by real-time quantitative polymerase chain reaction(RT-qPCR).The main results were as follows:1.Using iTRAQ technology,4071 proteins were identified from ‘Ganqi 3' and‘Newhall' navel orange during fruit development(60 days after flowering,150 days after flowering,210 days after flowering).The number of DEPs(fold change ?1.2,p ?0.05)between the two varieties is 149 which included 89 up-regulated proteins and 60down-regulated proteins.The bioinformatics analysis showed that the most of DEPs significantly enriched in thylakoid and photosystem terms under cellular component ontology;chlorophyll binding,tetrapyrrole binding and chromatin binding terms under molecular function ontology;photosynthesis,generation of precursor metabolites and energy,light reaction of photosynthesis terms under biological process ontology;and pathways such as photosynthesis,metabolic pathways,photosynthesis-antenna proteins,phenylpropanoid biosynthesis and synthesis of secondary metabolites.2.Using iTRAQ technology,6749 proteins were identified from ‘Ganqi 3' and‘Newhall' navel orange at three cold storage period(0 days,90 days,150 days).The number of co-expressed proteins in two biological replicates were 4040,the number of DEPs(fold change ?1.2,p ?0.05)at three cold storage stages were 610(265 up-regulated and 345 down-regulated),1262(630 up-regulated and 632 down-regulated),307(93up-regulated and 214 down-regulated)respectively.From all three storage period stages,a total of 127 DEPs were screened,including 47 up-regulated proteins and 80down-regulated proteins.The bioinformatics analysis showed that the most of DEPs main significantly enriched in cell wall,external encapsulating structure,plant-type cell wall under the cellular component ontology;the hydrolase activity,glycosyl bonds,hydrolase activity,hydrolyzing O-glycosyl compounds,cation binding,C4-dicarboxylate transmembrane transporter activity,lipoxygenase activity under molecular function ontology;S-adenosylmethionine metabolic process,C4-dicarboxylate transport,oxylipin biosynthetic process,carbohydrate transport under biological process ontology;andpathways such as phenylpropanoid biosynthesis,other glycan degradation,linoleic acid metabolism,starch and sucrose metabolism,cyanoamino acid metabolism,biosynthesis of secondary metabolites and so on.3.The combined analysis of proteomic and previous transcriptome data showed that the number of associated proteins with transcriptome data during fruit development is 2903 with the correlation coefficient of-0.0170.A total of 35 significantly DEPs are related to the differential expression genes(DEGs)revealed by transcriptome,of which 11 DEPs(9up-regulated,2 down-regulated)had the same expression trend as DEGs revealed by transcriptome with the correlation coefficient of 0.4091,While 24 DEPs had the opposite trend with correlation coefficient of-0.6739.The correlation coefficients of DEPs related to DGEs at three cold storage stage quantitative protein were 0.0988,0.1606,0.0881 respectively.The related DEPs number and correlation coefficients of three cold storage stages were 6/0.8286,37/0.5139 and 15/0.6857 respectively.The correlation coefficient of DEPs with the same expression trend of three cold storage stages were 0.8286,0.7475 and0.6978,while with the opposite trend were 0,-0.2167 and 1.4.Based iTRAQ and bioinformatics analysis results,some pathways which may affect the wax synthesis and transport and storage properties were screened out including Cutin,suberin and wax biosynthesis,Diterpenoid biosynthesis,Fatty acid biosynthesis,Fatty acid elongation,Terpenoid backbone biosynthesis,Sesquiterpenoid and triterpenoid biosynthesis,ABC transporters,Plant hormone signal transduction,Plant-pathogen interaction.One hundred and sixty-seven DEPs were screened from these pathways.5.Twenty-seven DEPs were selected to perform RT-qPCR verification.The results showed that 11 DEPs had the same expression trend at both mRNA level and protein level,while 6 DEPs had the opposite trend.In addition,several key DEPs including Cschlp/bchp,CsABCG2,CsFAR,CsHHT1,CsfabG-1,CsTPS1,CsABCG2-1,CsABCG2-2,CsKAR,CsMYC2,CsGID1,which had the consistent expression trend at both mRNA level and protein level were screened in this study.6.Combined with iTRAQ analysis,proteomics and transcriptome correlation analysis,RT-qPCR analysis and the difference of fruit peel wax contents between ‘Ganqi 3' and‘Newhall',we speculated that fruit development protein CsABCG2(Cs6g15280)may be the key protein for a glossy phenotype of ‘Ganqi 3';cold storage proteins CsFAR(orange1.1t02246),CsABCG2-1(Cs6g15300),CsKAR(Cs7g09930),CsCER10(Cs7g29210),CsHHT1(Cs2g05090),CsTPS1(Cs5g12900),CsMYC2(Cs3g17300)and CsPR1(Cs8g03430)might be the key proteins which may direct or indirect effect the fruit peel wax contents and cold storage properties of ‘Ganqi 3' navel orange.