Molecular Mechanism Of Fruit Ripening Mediated By PpHB.G7 And PpNAC.A18

Prunus persica((L.)Batsch.),one of the native fruit in China,has planted in large areas around the world.The peach fruit is brightly colored,the pulp is crispy,tender and juicy,the flavor is beautiful,and the nutritional value is very high,so it is very popular among consumers.Peach fruit is a climacteric fruit,and the maturity refers to the process in which the fruit undergoes a series of physiological and biochemical changes at the end of growth and reach the best state of the edible variety.The ripening peach fruit show the soften of the flesh,high juice concentration,change the outer color and increase ethylene content.There are a large release of ethylene during fruit ripening,and the fruit matures quickly and has a short shelf life.Therefore,depth study of the molecular mechanism of peach fruit ripening has important guiding significance to regulate fruit ripening process,and it also can improve peach fruit quality,effectively maintain postharvest quality and prolong storage time.In this paper,RNA-seq analysis of peach fruits in different developmental and mature stages was used to screen for genes involved in fruit maturation,and through transient expression,Dual Luciferase Reporter gene assay(DLR),Yeast one-Hybrid(Y1H),Electrophoretic Mobility Shift Assay(EMSA),genetic transformation and other experimental methods have studied the mechanism of ERF,HB and NAC transcription factors regulating peach fruit ripening.The conclusions of this thesis are as follows:1.Transcriptome analysis of fruit samples of ’Nanshan Sweet Peach(NS)’ and’Zhaohui(ZH)’ in four different stages.The results showed that there were 176 upregulated candidate genes in fruit ripening,and further test performed with qRT-PCR in twelve peach cultivars,the results showed ACS,ACO,ERF,NAC,HB and other gene families related to peach fruit ripening.Further identification and expression analysis of ACS and ACO genes in peach showed that PpACS.Al(Ppa004774m)and PpACOA1.1(Ppa008791m)were involved in the ripening process of peach fruit.2.RNA-Seq analysis and family identification of ERF in peach fruit showed that PpERF.A16 gene was involved in fruit ripening.Peach fruit was treated with ethephon and 1-MCP.Testing the changes of fruit firmness and the expression of related genes were analyzed by qRT-PCR.The results showed that exogenous ethylene can induce the expression of PpERFA16,PpACO.A1.1,PpACS.A1 and PpEndoPGM genes.In order to verify the function of PpERF.A16 gene,transient transformation was carried out,the results indicated that PpERF.A16 is involved in ethylene synthesis and conduction pathway by affecting the expression levels of PpACO.A1.1,PpACS.Al and PpEndoPGM genes.The dual luciferase reporter assay showed that PpERF.A16 had a regulatory effect on the promoters of PpACS.A1,PpACO.A1.1 and PpEndoPGM.Further validation and clarification of PpERF.A16 by Yeast Single-Hybrid(Y1H)and Electrophoretic Mobility Shift Assay(EMSA)can directly regulate ethylene synthesis and conduction pathways through binding to GCC-box of PpACS.A1 and PpACO.A1.1 promoters.3.By RNA-seq and qRT-PCR analysis in peach fruit,it was found that HD-ZIP Ⅱmember PpHB.G7 may be involved in the fruit ripening process.Transient expression analysis of PpHB.G7 in peach fruit by overexpressing and silencing this gene showed that the overexpression of the gene lead an increase in ethylene release and a significant increase in the expression of PpACS.A1,PpACOA1.1 gene in peach fruits,while the silencing of the gene resulted in the opposite result.Furthermore,the relative ratio of LUC/REN was detected by Dual-Luciferase Reporter Assay and verified by Yeast one-Hybrid.These results suggested that PpHB.G7 regulated ethylene biosynthesis by binding to the promoters of PpACS.A1,PpACO.A1.1 and then participated in the regulation of fruit ripening.4.Overexpression and silence of PpNAC.A18 gene and transient expression analysis of PpNAC.A18 gene showed ethylene production was increased and the expression of PpACO.A1.1,PpACS.A1,PpEndoPGM were higher significently.And silencing the PpNAC.A18 gene had the opposite results.So PpNAC.A18 was involved in the regulation of ethylene synthesis and fruit ripening by affecting the expression levels of PpACO.A1.1,PpACS.A1,PpEndoPGM and PpERF.A16 genes.The Dual Luciferase Reporter assay showed that only co-transformed pCAMBIA1301:NAC.A18 and PpERF.A16pro-LUC/CaMV35S-REN had higher relative value of LUC/REN than co-transformed PpERF.A16pro-LUC/CaMV35S-REN and empty vector,this result indicated that PpNAC.A18 had a regulatory effect on the promoter of PpERF.A16 gene,but had no significant regulatory effect on the promoters of PpACS.A1,PpACO.A1.1 and PpEndoPGM.Furthermore,it was confirmed by Yeast one-Hybrid(Y1H)that PpNAC.A18 can bind to the promoter of PpERF.A16 gene,and then regulate ethylene synthesis and signal transduction pathway.