| NAP(NAC-like,activated by AP3/PI)subfamily belongs to a member of the NAC transcription factor,which is considered to be related to plant specific transcription factors that control plant leaf senescence,regulate plant growth and development,and respond to abiotic stress.Previous studies identified seven NAP genes in the peach genome of which PpNAP4 and PpNAP6 showed significant expression during fruit ripening and softening.Therefore,in this study,the subcellular localization of PpNAP4 and PpNAP6 was performed using the ’RH’ test material;then the fruits of ’YH 11’ and ’MQ’ peach were treated with ethephon and ABA respectively,and the expression patterns of PpNAP4 and PpNAP6 under exogenous hormones were analyzed by RT-PCR.’RH’(melting)and ’JT 5’(nonmelting)were selected to analyze the differences expression of PpNAP4 and PpNAP6 in fruits of different textures.Secondly,using ’RH’ as experimental material,the functional studies of PpNAP4 and PpNAP6 were performed using virus silencing VIGS technology to further elucidate the roles of PpNAP4 and PpNAP6 in ethylene synthesis and metabolism.At the same time,’YSH’was used as a test material to construct an overexpression vector to assist in the study of the function of PpNAP4 and PpNAP6 in fruit ripening and softening,in order to explain the function of PpNAP4 and PpNAP6 in peach fruit ripening and its possible regulatory mechanisms.The soft melting peach variety was selected in the VIGS silencing experiment in order to make the experimental result more obvious,and the same as hard melting peach variety was selected in the same transient overexpression experiment.The main results are as follows:1、In the ’RH’ peach fruit,the GFP vector with the green fluorescent protein gene was linked to the target genes PpNAP4 and PpNAP6,and the subcellular localization of the protein in tobacco epidermal cells was performed by injection.The results showed that both PpNAP4 and PpNAP6 were located in the nucleus.2、In the process of ripening and softening of peach,the fruit of ’YH 11’ peach was treated with exogenous hormone ethylene and 1-MCP,Ethylene promoted fruit ripening,firmness decreased,ethylene release increased,and the expression levels of PpNAP4 and PpNAP6 were affected induced by ethylene and 1-MCP inhibited the fruit ripening;At the same time,exogenous hormones ABA and Flu were used to treat the fruit of ’MQ’ peach,which also promoted fruit ripening.ABA promoted fruit ripening,and firmness decreased and its ethylene release increased.But the expression levels of PpNAP4 and PpNAP6 were not significantly affected.3、Virus-induced PpNAP4 gene silencing,transcriptional expression PpNAP4 was obviously suppressed at the early stage of storage,the fruit hardness and ethylene release were also inhibited,and the ripening process of the fruit was delayed,while the expression of PpNAP6 was significantly lower than that of the control during the whole storage period,and the expression was suppressed.Genes related to mature softening,such as ethylene biosynthesis and metabolic related genes Pp ACO1,Pp ACS1,Pp EIN2 and Pp ETR2,were inhibited to varying degrees in the RNAi fruit of pre storage,and the amount of Pp PME3 and Pp PG21 related to cell wall metabolism related genes were also suppressed during the whole storage period.During the whole storage,the adhesiveness of PpNAP4 silenced fruit was lower than that of the control fruit,while the springiness,cohesiveness,chewiness and gumminess were significantly higher than those of the control.The results showed that the PpNAP4 gene directly or indirectly involved in the related pathways of mature softening,and influenced the change of fruit texture,and played an important role in the ripening and softening process of peach fruit.4、Overexpression of PpNAP4 gene,the firmness and ethylene release of peach fruit were obviously promoted,and the process of maturation and softening was accelerated.The expression of PpNAP4 and PpNAP6 reached the peak value at the time of storage 6d,and the expression amount of PC1301-NAP4 fruit was significantly higher than that of the control at the time of overexpression,but expression in other periods was generally the same as or lower than the control.overexpressed the PpNAP4 gene,the change of texture was opposite to the change of the essence of the silencing PpNAP4,in which the adhesiveness was higher than the control fruit,and the other indexes were generally lower than the control fruit.This result fully indicates that PpNAP4 has an important effect on fruit texture and plays an important role in fruit ripening and softening. |
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