The Function Analysis Of CmNAC56 Gene In Melon Fruit Ripening Process

Muskmelon（Cucumis Melo）is a creeping vine,and a dicotyledonous diploid plant that belongs to the Cucurbitaceae Muskmelon cultivation has a long history in China,and the planting area is broad.Its fruit has rich nutrients and trace elements,which make it popular among people.In recent years,the fruit of muskmelon has played a significant role in the study of the ripening mechanism of fleshy fruit.In this study,we studied the function of the CmNAC56 gene differentially expressed in fruit development was explored by analyzing transcriptome data using Cucumis melo L.cy Hetao which owns excellent quality and traits as experimental material.The main results are as follows:（1）The bioinformatic results of CmNAC56 in melon demonstrated that was located on chromosome 6 of melon.Besides,CmNAC56 encoded 353 amino acids,the protein products were all stable hydrophobic whose molecules were39371.84 Da and the theoretical isoelectric point is 8.32.These proteins had no precursor peptide,no signal peptide,and no transmembrane domain.Its secondary structure was mixed,consisting ofαhelix,βfolding and random coil.There was 7 protein,including AP2-like ethylene-responsive transcription factor TOE3,were predicted to interact with CmNAC56 directly.（2）Employing the muskmelon genome database,we found that the c DNA length of the CmNAC56 gene was 1678 bp,including 3 introns and 3 exons.The open reading frame sequence of CmNAC56 cloned using the designed primer was 1062 bp.And the overexpression vector p PZP221（35S-CmNAC56-NOS）and gene editing vector p YL-CmNAC56 were constructed successfully.（3）Through Agrobacterium-mediated transient expression technology,Agrobacterium tumefaciens solution containing overexpression vector p PZP221（35S-CmNAC56-NOS）was injected into early ripening melon fruits with a syringe for transient expression.Through observation and analysis,the parts without injection of bacterial liquid developed normally,and the color of the exocarp of the injection site was delayed to turn yellow.All these suggested that the overexpression of the CmNAC56 gene might delay fruit ripening to some extent.（4）Overexpression vector p PZP221（35S-CmNAC56-NOS）and gene editing vector p YL-CmNAC56 were introduced into muskmelon using ovary injection.As a result,the positive rates of T1 transformed seeds generation detected by PCR were 10.8%and 9.2%,respectively and the ones with high positive rates were sown in the field.By observing and recording the maturity of T2 generation fruits,it was found that the fruits of the transformed plants with CmNAC56 gene editing ripened 2-3 days earlier than those of the wild type,while the fruits of the transformed plants with CmNAC56 overexpression ripened 2-3 days later than those of the wild type.（5）The endogenous ethylene content of T2 generation positive fruits was analyzed after 30 d,35 d,and 40 d of fruit development.It was found that the ethylene content of overexpressed fruits was higher at 35 d,and there was no significant difference between the ethylene content of gene-edited fruits and untransformed fruits.In the T3 generation seeds,the full granules were randomly selected to carry out the"triple response"experiment,it turned out that the"triple response"effect of gene-edited seedlings>control group seedlings>gene overexpressing seedlings.q RT-PCR was performed on the edited fruits of different periods,and the results suggested that both target sites1 and 2 were expressed,and Cas9 was expressed.And we also found that the relative expression levels of ethylene--responsive transcription factors ERF24and ERF61 were higher in the gene-edited fruits,and the relative expression levels of ethylene synthesis related genes ACO1 and ACO4 were higher in the overexpressed fruits.The relative expression level of the CmNAC56 was higher in edited fruits and lower in overexpressed fruits.q RT-PCR results also told us that in roots of seedlings treated with different concentrations of Cu²⁺,Fe²⁺,and Mn²⁺,respectively,the promotion effect was obvious when the ion concentration is 50 m M,and the effect of Fe²⁺is the greatest while Cu²⁺and Mn²⁺have no obvious rule.To conclude,CmNAC56 plays something in muskmelon fruit development,like promoting lateral root growth during development,and this gene was at least partially ethylene-dependent expression or may-be a downstream regulator of ethylene.