Cloning And Expression Analysis Of AP2/ERF Related To The Formation Of Aromatic Apocarotenoids In Apricot Fruits

Apricot(Prunus armeniaca L.)is an important stone fruit tree,Xinjiang apricot is widely prefered by consumers in the market due to its unique flavor,and can also be processed into fresh fruit juice,preserved fruit,jam and so on.Fruit quality is mainly composed of color,texture and flavor,the flavor quality is determined the combination of the taste and aroma,but the aroma is generally considered to play the crucial role in the whole flavor formation.As a plant-specific transcription factor,AP2/ERF directly or indirectly involve in the formation and regulation of ripening,texture,flavor and color in many fleshy fruits.Our previous study showed that apricots are rich in aromatic apocarotenoids,however,it is still unknown whether the AP2/ERF transcription factors involve in the formation of aromatic apocarotenoids in apricot fruits.In this study,we detected the composition and content of aromatic apocarotenoids from five Xinjiang apricot cultivars during development and ripening by Gas Chromatography-Mass Spectrometry(GC-MS).Based on ‘Jianali' transcriptomics data,Weighted Gene Co-expression Network Analysis(WGCNA)was used to screen the specific AP2/ERF transcription factor members related to the formation of the aromatic apocarotenoids,and then the full-length sequences were cloned and their functions were charcterised putatively by bioinformatical analysis.Finally,the specific AP2/ERF members involving in the aromatic apocarotenoid in apricot fruit were ascertained by gene expression pattern analysis and correlation analysis.The main results are as follows:1.A total of four aromatic apocarotenoids were identified from fruit of the five cultivars(‘Baixing',‘Lajiaoxing',‘Shushanggan',‘Jianali' and ‘Danxing'),including ?-ionone,dihydro-?-ionone,6-methyl-5-hepten-2-one and ?-damascenone,of them,?-ionone is the most abundant apocarotenoid,followed by dihydro-?-ionone,and ?-damascenone.The richest contents of the four aromatic apocarotenoids were detected in ‘Baixing' fruit among the all cultivars tested.The contents of all apocarotenoid increased with fruit ripening process.2.WGCNA and network visual analysis demonstrated that four ethylene transcription factors AP2/ERF were suggested the putative regulatory role in the formation of the aromatic apocarotenoids during apricot fruit ripening,namely,PaERF4(Unigene16935),PaERF12(Unigene3832),PaERF26(Unigene8329)and PaAP2-like(Unigene 18939).3.The full length of target genes were cloned by RACE technology,the basic bioinformatic analysis showed that the AP2/ERF protein sequence contains at least one AP2 conserved domain,and the AP2 domains of PaERF4 and PaERF12 also contains a transcription suppression EAR-motif,indicating that PaERF4 and PaERF12 may play the negative regulatory role in the formation of the aromatic apocarotenoids in apricot fruit during ripening.4.The expression patterns of AP2/ERF gene and structural genes CCDs and NCEDs responsible for the biosynthesis of aromatic apocarotenoids were analyzed using qRT-PCR,the results showed that PaERF4 and PaERF26 presented the down-regulated gene expression pattern during ripening,while PaERF12 presented the opposite gene expression pattern in ‘Baixing' and ‘Jianali' cultivars,and PaAP2-like gene expression was up-regulated.Except for PaNCED1(U19678),other related genes(PaCCD1,PaCCD4 and PaNCED)were up-regulated during the ripening process.5.The correlation analysis between carotenoids and apocarotenoids with AP2/ERF genes and structural genes showed that the expression of AP2/ERF genes and structural genes correlated with the carotenoids and apocarotenoids production.In the ‘Baixing',the expression levels of PaERF12 and Pa ERF26 showed positive correlation with the contents of the apocarotenoid,but their correlations were different in the ‘Jannali'.The expression of PaERF4 negatively correlated with the main apocarotenoid content in two apricot fruit cultivars,but the gene expression of PaAP2-like positively correlated with the content of main carotenoids and apocarotenoids in ‘Baixing',but negatively correlated in 'Janali'.In summary,the ethylene transcription factor PaERF4 was inferred to play the negative regulatory role in the formation of aromatic apocarotenoids in apricot fruit during the development and ripening,but PaERF12,PaERF26 and PaAP2-like genes have different regulatory effect on the formation of aromatic apocarotenoids and their affects might depend on cultivars.