Cloning And Function Characterization Of Genes And Their Promoters Of Two Carotenoid Cleavage Dioxygenases From Apricot Fruit

Apricot(Prunus Armeniaca L.)is oneofan important fruit tree that belong to the family Rosaceae.Its fruit was popularin the fruit market with consumers owing to its bright color,uniqueflavor andabundant nutrients.As a unique ecological varietal group,Xinjiang apricot has many variation typesforcolor,such as white,yellow,orange and red,and its background color is mainly decided by the carotenoids accumulation.Our previous study found that carotenoid cleavage dioxygenase gene(CCD)plays an important role in the color formation and aroma apocarotenoidsbiosynthesis of apricot fruit,but its specific role and mechanism is not clear.In this study,based on two CCDs identifiedfrom the transcriptomics,Xinjiang apricot fruits with different color were used asmaterial,the composition and content variation of carotenoids and aroma apocarotenoidsduring fruit ripening were detected by high performance liquid chromatography(HPLC)and gas chromatography-mass spectrometry(GC-MS),respectively.The roleof PaCCD1 and PaCCD4 in the carotenoids metabolism forapricot fruit was analyzedby means of gene expression pattern analysis,gene cloning,subcellular localization,bioinformatics analysis and promoter deletion.The main results are as follows:1.A total of seven carotenoids were detected from the fruits of the tested cultivars,including?-carotene,?-cryptoxanthin,?-carotene,phytoene,neoxanthin,xanthophyll,and violaxanthin.The dark-colored varieties(Dan apricot and Hongyu apricot)are mainly contain ?-carotene,which accounted for 64.09% and 73.32% of the total carotenoids content in the peels,respectively,and also contain ?-carotene and ?-cryptoxanthin,xanthophyll,violaxanthin and a small amount of neoxanthin,among which ?-carotene and ?-cryptoxanthin were detected only in the two dark-colored varieties,while the content of carotenoids in the light-colored varieties(Luntaixiaobai apricot and White apricot)was significantly lower than that in the dark-colored cultivars.Luntaixiaobai apricot was mainly contain xanthophylls,which accounted for68.89% of the total carotenoids content in the peels.White apricot mainly accumulates xanthophylls and ?-carotene,which accounting for 38.94% and 24.87% of the total carotenoids content in the peel,respectively.During fruit development,the main carotenoids in the dark-colored varieties rapidly accumulated,while the content of ?-carotene in the light-colored varieties rapidly decreased and the content of phytoene increased during fruit ripening.Three kinds of aroma apocarotenoids,including ?-damascone,?-ionone and dihydro-?-ionone,were detected from tested fruits,of which the ?-ionone was the most abundant.During fruit development,the aromaapocarotenoids in fruits of all cultivars increased significantly,and the content of ?-ionone in light-colored varieties was significantly higher than that in dark-colored cultivars.2.The full-length sequences of PaCCD1 and PaCCD4 genes were cloned from the fruit of Luntaixiaobai apricot using rapid-amplification of cDNA ends(RACE)method.The open reading frame(ORF)s of PaCCD1 and PaCCD4 were 1644 bp and 1812 bp,encoding 547 and 604 amino acids respectively.The molecular weights of PaCCD1 and PaCCD4 proteins were 61.699 kD and 66.021 kD,and the isoelectric points were 6.16 and 6.65,respectively.Phylogenetic analysis showed that both genes could cluster together with the corresponding gene family members with known functions,and had the highest homology with Rosaceae peach.The protein structure analysis showed that they both contain typical RPE65 domains,which are consistent with the characteristics of the CCD family members.Subcellular localizations were performed for the two genes,showing that PaCCD1 is localized in the cytoplasm whereas PaCCD4 is localized in the plastids.3.The temporal and spatial expression patterns of PaCCD1 and PaCCD4 were analyzed by real-time fluorescence quantitative(qPCR).It was found that PaCCD1 and PaCCD4 had the highest expression in fruits,but also in flowers,and they were basically not expressed in roots,stems and leaves,showed that CCDs are specifically expressed in fruits.The expression level of PaCCD1 was increased in the first four development stages of all cultivars,which was consistent with the change trend of aroma apocarotenoids in fruits,indicating that PaCCD1 may be involved in the degradation of carotenoids in the apricot fruit to form aroma apocarotenoids.The expression level of PaCCD4 gene was higher in light yellow cultivar than in red-orange cultivar,indicted that the yellow fruit has lighter color due to the higher expression of the PaCCD4 and the red-orange fruit has darker color due to the lower expression ofPaCCD4,also indicating that PaCCD4 may be involved in the regulation of carotenoid accumulation in apricot fruits.However,the carotenoid metabolism-related genes PDS,CRTISO and ZEP had little effect on carotenoid regulation during fruit development,while PSY,LCYB and CHYB all had certain regulatory effects on the carotenoid synthesis in fruits.4.In order to find the upstream regulatory elements of the PaCCD1 and PaCCD4 genes in apricot fruit,we used the genome walking method to clone the gene promoter regionsof PaCCD1 and PaCCD4 with 2233 bp and 1838 bp sequences,respectively.Cis-acting elements analysis revealed that both promoters contain multiple cis-acting elements which interact with light,abscisic acid,methyl jasmonate,and other environmental factors,hormone signals,and regulatory tissues.In addition,the PaCCD1 promoter contains stimulus-inducing and heat-stress response elements.The PaCCD4 promoter contains elements of gibberellin,low temperature and enhanced transcription levels,indicating that the CCDs gene family can participate in a variety of life activities during plant development.5.Withaiming to determine the core region of the gene promoter,a promoter deletion assay was used to construct a 5'-Endpromoter deletion fragment and transiently transform tobacco to perform GUS activity detection.The results showed that the activity of GUS of PaCCD1 and PaCCD4 was gradually weakened with the deletion of the promoter fragment.The core region of the PaCCD1 promoter was within the 600bp-2233 bp region,and the core region of the PaCCD4 promoter was among the 600bp-1838 bp region,which lays a foundation for the further screening of transcription regulatory factors.