Functional Analysis Of VvERF17 Regulating Chlorophyll Degradation In Grape Berry

The color of grape(Vitis vinifera L.)peel is an important indicator of fruit quality.The development of grape fruit is accompanied by the accumulation of anthocyanins and the degradation of chlorophyll,so the chlorophyll in the peel will affect the color of the fruit.Grape ethylene response factor(VvERF17)which is a potential transcription factor that may affect the degradation of grape skin chlorophyll was selected as the research object.This study first predicted a transcription factor VvERF17 that may be related to the degradation and metabolism of grape skin chlorophyll through homologous sequence alignment.Ethylene treatment experiments proved that the expression of VvERF17 is highly correlated with the degradation of grape peel chlorophyll.Subsequently,a series of experiments were conducted to verify the interaction between the VvERF17 protein and the promoter of chlorophyll catabolic gene(CCG).Finally,the plant transient transformation system clarified the molecular mechanism of VvERF17 regulating the degradation of chlorophyll in grape skin.The main findings are as follows:1.VvERF17 was identified by homologous sequence alignment.VvERF17 protein contains AP2 domain,which is the DNA binding site of plant AP2 and EREBPs protein.Phylogenetic analysis showed that the phylogenetic relationship is very close between VvERF17 and Cit ERF13,which regulates peel chlorosis in citrus.The VvERF17 coding sequence was cloned using ‘Kyoho’ grape c DNA as a template.2.The expression pattern of VvERF17 in 6 grape varieties was detected by q RT-PCR.The result showed that VvERF17 was highly expressed in leaves and fruits,in addition,the expression level of VvERF17 in the skins of 6 grape varieties is closely related to the coloration of grape skins.The expression trend of major CCGs(Vv NOL,Vv PPH,Vv PAO and Vv RCCR)was similar to that of VvERF17,and was negatively correlated with the change of chlorophyll content.After treating the clusters of six grape varieties with ethylene donor ETH and ethylene inhibitor 1-MCP,VvERF17 was increased in response to ethylene signal,and the co-expression of CCGS and VvERF17 was observed,which suggesting that VvERF17 was regulated by ethylene signal,the degradation of chlorophyll in grape skins was closely related to the expression intensity of VvERF17 and CCGS;3.VvERF17 protein is localized in the nucleus and has strong transcriptional activation activity.Vv NOL,Vv PPH,Vv PAO and Vv RCCR are all target genes of VvERF17.VvERF17 directly binds to the cis-acting elements of the promoters of these 4 CCGs and activates the transcription of downstream genes;4.Heterogeneous transient transformation was carried out in tobacco leaves,tomato fruits and strawberry fruits,and homologous transient transformation and RNAi experiments were carried out in grape leaves.Finally,a working model of ethylene regulating the chlorophyll degradation of grape peel was constructed: VvERF17 was combined with The promoter cis-acting elements of CCGs activate their transcription and coordinately regulate key genes in the chlorophyll degradation pathway to promote chlorophyll degradation.These results explain the working mechanism of chlorophyll degradation in grape skins and enrich the understanding of the color formation of grape skins.