Study On The Molecular Mechanism Of 1-MCP Treatment To Cause The Ripening Disorder Of Papaya

Papaya（Carica papaya L.）is one of the important economic crops in the subtropics of China,with high nutrition and health value.Papaya is a typical respiratory jump type fruit.It matures quickly after harvest,is easy to soften and rot,and is not easy to keep fresh.It is seriously lost during storage and transportation.Ethylene receptor inhibitor1-methylcyclopropene（1-MCP）can effectively delay fruit ripening and softening,but inappropriate 1-MCP treatment can easily cause papaya fruit not to soften and cause hind-ripening obstacles.Therefore,the study of the molecular mechanism of 1-MCP causing late ripening obstacles has important guiding significance for the application of papaya storage and preservation technology.In this study,the phenotype of papaya obtained by the research group in the previous period of 1-MCP treatment was used as the research basis.The content of IAA during the maturation process after different treatments was measured by liquid-mass spectrometry.And mining,through RT-q PCR gene expression verification,a large number of differentially expressed transcription factors and differential functional protein genes were screened,Ethylene response factor CpERF WRI1 and MADS transcription factor CpAGL18,the most significant differential expression factor,were used as the main research objects.Through dual-luciferase reporter gene experiment（Dual-luciferase）,both auxin signaling pathway and ethylene signaling pathway may be explored Target genes and regulatory effects,and analysis and verification of transcriptional regulatory mechanisms,with a view to elucidating the molecular mechanism of unsuitable 1-MCP treatment after ripening barrier The main research work and results are as follows:1.The auxin content of papaya papaya treated with CK,1-MCP treatment for 16h and1h was analyzed,and it was found that the dynamic change of IAA was negatively correlated with the fruit softening disorder caused by 1-MCP treatment.After the 1-MCP treatment of papaya fruit for 16 h,the auxin content was suppressed at a very low level during the entire post-ripening process.After 1 h treatment with 1-MCP,the auxin content did not change much compared with the control,and the peak appeared On the 2nd day,it was delayed by 1 day from the control.The fruits treated with 400n L·L^-11-MCP combined with 2000n L·L^-1NAA can alleviate the post-maturity barrier,and the fruits can be ripened and softened.2.Based on proteome differential protein analysis,17 new differential proteins were screened.RT-q PCR results showed that the gene expression levels of CpGDH2,CpUCK,CpTrp B,CpMST,and CpSAM were positively correlated with late maturity,and were inhibited by 1-MCP,and 1-MCP16h Completely inhibit the expression of CpGDH2 and CpUCK.It is speculated that CpGDH2,CpUCK,CpTrp B,CpMST and CpSAM are the key differential proteins in the post-maturation disorder caused by 1-MCP.3.Based on the analysis of transcriptome differential genes,13 ethylene and auxin signaling pathway genes closely related to maturity and 8 MADSs transcription factors were screened.RT-q PCR results showed that CpERF WRI1,CpERS1,CpAGL18,CpAGL19,CpIAA16-like,CpGH3.1,CpARG7,CpSAUR21-like,CpSAUR32-like,CpARF2,CpAuxin-induced protein6B are all positively correlated with post-maturing,the expression level is significantly inhibited by 1-MCP.CpAGL30,CpARF19-like are two negatively correlated with post-maturing Transcription factor,expression level was significantly induced by 1-MCP.4.Based on the transcriptome sequencing library,CpAGL18 and CpERF WRI1full-length cDNA sequences were cloned,and the cDNA sequences were 777 bp and 1176bp,respectively encoding 259 and 392 amino acids,the corresponding protein molecular weights were 29.54 k Da,44.56 k Da,and the isoelectric point was 8.82,5.18.Amino acid sequence alignment and phylogenetic tree analysis showed that CpAGL18 has M,I,C,and K domains and belongs to the typical MADS family protein,which is closely related to At AGL18.CpERF WRI1 belongs to the AP2/ERF transcription factor family and is closely related to Md ERF WRI1.Subcellular localization results show that CpAGL18 is localized in the Nucleus and cell membrane,and CpERFWRI1 is localized in the nucleus,which is a nuclear protein.5.Using yeast two-hybrid and BIFC technology,the interaction relationship between CpAGL18 and CpEBF1/2 was determined.6.Dual-luciferase reporter gene experiment（Dual-luciferase）shows that CpAGL18can activate the activity of CpWAT1 and CpAuxin-inducedprotein6B promoters to promote their expression,CpAGL18 and CpEBF1 interact more significantly to promote the promoter activity of CpWAT1,CpERF WRI1 can be activated The promoter of CpERS1.Yeast One-Hybrid Assay analysis further showed that CpAGL18 can bind the CpWAT1promoter,and CpERF WRI1 can bind the CpERS1 promoter.Papaya leaf transient expression experiments showed that CpERF WRI1 can affect the gene expression level of CpERS1.In summary,it is speculated that the inappropriate 1-MCP treatment significantly inhibited the gene expression of CpAGL18 and CpEBF1 during the post-ripening process of papaya.On the one hand,CpAGL18 and its interaction protein CpEBF1 suppressed the gene expression level of CpWAT1 through transcriptional regulation,which significantly affected The auxin content affects the ripening and softening of the fruit;On the other hand,by inhibiting the expression of CpERF WRI1,which in turn affects the expression of CpERS1,affects the ethylene signal transmission and the production of ethylene in System II,thereby further suppressing the ethylene reaction and triggering the fruit ripening disorder.