The Preliminarily Functional Analysis Of Melon CmNADP-ME3 And CmDFR11 Genes In Fruit Development

Melon belongs to Cucurbitaceae and melon genus,it is also an annual dicotyledon herb.In this study,CmNADP-ME3 and CmDFR11 genes were selected as candidate genes,by retrieving the transcriptome data of melon completed in the early stage in our laboratory,which are high expression in melon fruit ripening stage and respiratory climacteric stage.Bioinformatics analysis and real-time fluorescence quantitative PCR were used to analyze the expression levels of two candidate genes in different developmental stages of melon fruit and different tissues and organs of melon.Cloning and construction of superexpression vector and CRISPR/Cas9knockout vector,then transfer the constructed vector into melon by ovary injection method.The main results are as follows:1.Six members of CmNADP-ME gene family and fourteen members of CmDFR gene family were identified from Melon genome by bioinformatics method.The promoter elements of CmNADP-ME3 and CmDFR11 genes were analyzed,it was found that the promoter of CmNADP-ME3 gene contained abscisic acid,jasmonic acid,ethylene,salicylic acid,auxin and gibberellin response elements,the promoter of CmDFR11 gene contains abscisic acid,salicylic acid,auxin and gibberellin response primitives.Chromosome location analysis revealed that CmNADP-ME3 was on chromosome 3 and CmDFR11 was on chromosome 7.2.Real-time fluorescence quantitative PCR analysis showed that the expression of CmNADP-ME3 gene in melon fruit increased rapidly at maturity and reached the highest level at respiratory climacteric period,and the expression level in the respiratory climacteric period was significantly higher than that in the root,stems and leavess（P-value<0.01）,which were 51,9.2 and 32 times higher than that in the roots,stems and leaves,respectively.The expression level of CmDFR11 gene in melon fruit increased rapidly at maturity and reached the highest value at maturity period,and the expression level in mature period was significantly higher than that in roots,stems and leaves（P-value<0.01）,which were 40,40 and 15.6 times higher than that in roots,stems and leaves,respectively.3.The full length of CmNADP-ME3 open reading frame 1770 bp and CmDFR11open reading frame 1059 bp were cloned successfully by RT-PCR.4.The results of stable genetic transformation showed thatoverexpressed of CmNADP-ME3 in T₁ melon matured 4-6 days earlier than the control fruit,and the overexpression of CmNADP-ME3 in T₁ fruit was 5 times as much as that in control fruit.Overexpression of CmDFR11 in T₁ melon matured 4-6 days earlier than the control fruit,and the overexpression of CmDFR11 in T₁ fruit was 3.08 times as much as that in control fruit.All the above results proved that the expression of CmNADP-ME3 and CmDFR11 could promote melon fruit ripening.