Screening And Expression Analysis Of Differentially Expressed Genes Based On Transcriptome Data During Vaccinium Corymbosum Early Fruit Development

Fruit size is an important agronomic trait of blueberry(Vaccinium spp.),which is related to cell proliferation and cell expansion.The early stage of fruit development is the key period to determine blueberry fruit size.In this study,southern highbush blueberry cultivars 'O'Neal' and 'Bluerain' during early fruit development(S0-S2)were used for transcriptome sequencing and differentially expressed genes(DEGs)screening.Then partial DEGs were isolated,analyzed its expression patterns,and identified its biological functions by virus-mediated gene silencing(VIGS).These results will help us to understand the molecular mechanisms during early blueberry fruit development,and the key genes regulated fruit size during early development.The specific results were as follows:(1)Transcriptome data analysis:(1)According to PCA anaylsis,our transcriptome data were reliabled,and the data of 'O'Neal' and 'Bluerain' were similar between stage S1 and S2.(2)45,474 DEGs were detected at the stage S0 from 'O'Neal' and 'Bluerain',including 20,827 up-regulated genes and 24,647 down-regulated genes;69,294 DEGs were detected at the stage S1,including 35,074 up-regulated genes and 34,220 down-regulated genes;69,816 DEGs were detected at the stage S2,including 34,027 up-regulated genes and 35,789 down-regulated genes.The results showed that more DEGs were existed in the samples of stage S1 and S2 than stage S0,indicated that stage S1-S2 were the key stages to determine early fruit development and fruit size.(3)GO enrichment analysis showed that the DEGs were mainly enriched in molecular function at the same stage of 'O'Neal' and 'Bluerain'.KEGG enrichment analysis indicated that DEGs were achieved through multiple metabolic pathways.Then 31 candidate genes were chosed by venn method to use for further analysis.(2)Real-time fluorescence quantitative PCR(qPCR)validation: The results showed that the expression patterns of 31 DEGs were significantly different during 'O'Neal' and 'Bluerain' early fruit development,but consistanted with transcriptome data.(3)Gene isolation and bioinformatic analysis: Three cytochrome P450-related genes and two auxin-related genes were isolated from the S1 stage of 'Bluerain' fruit by using homologous cloning method,including Cluster-24679.305304(VcCYP749A22),Cluster-24679.356572(VcCYP81E8),Cluster-24679.62988(VcCYP76A2),Cluster-24679.344142(VcILR1),Cluster-24679.308496(VcYUCCA10).The structures were relatively conserved with other plant species.(4)Gene function identification: Non-conserved fragments of seven differentially expressed cytochrome P450 genes were isolated,constructed into virus-induced gene silencing(VIGS)vectors,transformed into Agrobacterium tumefaciens GV3101 and infected green mature MicroTom fruit.10 days after infection,the color of VcCYP81E8(Cluster-24679.383750)-silenced fruit exhibited yellow,whereas the negative fruits became red,indicated that VcCYP81E8 gene might be involved in regulating carotenoid biosynthesis and metabolism.However,the biofunctions of VcCYP81E8 need to be further studied.