The Function Of Erf Transcription Factor Subfamily Genes During Melon Fruit Development

Melon is a world-wide cultivated economic fruit.Its economic value is primarily affected by the storability.Ethylene is one of the key factor responsible for ripening and storability.Although ERFs are a group of important transcription factors in the ethylene signal transduction pathway,little is known about the function of ERFs in the melon fruit development.In this study,the features of 64 ERF protein sequence and the promoter elements of these ERF genes in melon were analyzed by bioinformatics method.Cucumis melo L.cv.Hetao was used to study the function of melon ERF genes.The expression profiles of the ERF genes in different tissues were investigated using quantitative real-time PCR(qRT-PCR).Eight genes were identified as candidate genes related to fruit development.The functions of all the candidate genes were studied using a transient expression method.Furthermore,the overexpression,RNA interference(RNAi)expression and the gene editing expression vector were transformed into melon to authenticate its functions in the fruit development.The main results are as follows:1.The molecular weights of 64 ERF proteins were ranged from14.00 kDa to 46.62 kDa.The theoretical isoelectric points of forty proteins were less than 7.All the proteins are hydrophilic.With the exception of the CmERFIV-5 protein,all instability indexes of the ERF proteins were unstable;Fifty-eight proteins were localized in the nucleus,one was in the cytoplasm,and five were in the chloroplast.Twenty eight proteins contained signal-peptide-cleavage sites.A total of 83 promoter elements was predicted,including gibberellin,jasmonic acid,auxin,salicylic acid,abscisic acid,and ethylene response elements.Additionally,64 genes were unevenly distributed on 12 chromosomes.2.Real-time quantitative PCR was performed to detect the expression levels of the 64 CmERFs in melon root,stem,leaves,fruits at 9,18,27 and 36 DAP and fruits at climacteric as well as post-climacteric stages.The results showed that 8 genes were highly expressed at climacteric time.These 8 genes were differentially expressed at a significant level during climacteric and post-climacteric period.The expression of CmERFI-15,CmERFII-9,CmERFIV-2 and CmERFIV-3 at post-climacteric period was significantly higher than that in roots,stems and leaves.3.The functions of all the candidate genes were identified by the transient expression.The results showed that overexpression of CmERFI-15,CmERFII-9,CmERFIII-1,CmERFIV-2,CmERFIV-3 and CmERFIV-4 promoted fruit ripening,while RNAi of these genes inhibited fruit ripening;on the contrary,overexpression of CmERFI-18 and CmERFIII-13 postponed fruit ripening,while RNAi of these two genes accelerated fruit ripening.4.CRISPR-Cas 9 gene editing technique was conducted to verify the functions of CmERFI-15,CmERFII-9,CmERFIII-1,CmERFIV-2 and CmERFIV-3,which may play important roles in the fruit ripening,according to the results of transient expression.Moreover,the investigation of the maturations of T3 transformed fruits showed that,when compared with the wild type fruits,the ripening was advanced 7.58,7.47 and 6.98 days in three overexpression lines of CmERFI-15,respectively,while the maturations of RNAi fruits were delayed 9.20,10.60 and 11.08 days in this three transformed lines,respectively.CRISPR-Cas 9 gene edited fruits shared similar ripening period with RNAi fruits,which were delayed 13.23,14.63 and 14.53 days in three CmERFI-15 edited lines,comparing with wild type.We also found that the ripening was advanced 6.05,6.35 and 5.05 days in three overexpression lines of CmERF7II-9,respectively,while the maturations of RNAi fruits were delayed 8.15,8.30 and 9.10 days in this three transformed lines,respectively.CRISPR-Cas9 gene edited fruits shared similar ripening period with RNAi fruits,which were 11.80,12.40 and 12.75 days later in three CmERFII-9 edited lines than in wild type.5.Fruits with overexpressed CmERFI-15 at 28 DAP,RNAi fruits at 45 DAP,and gene editing fruits at 50 DAP were taken to determine the respiration rate.For CmERFII-9,overexpressed fruits at 29 DAP,RNAi fruits at 44 DAP,and gene edited fruits at 48 DAP were used to determine the respiration rate.It was found that the peak value of respiratory of overexpression fruits with CmERF1-15 and CmERFII-9 occurred at 18.24 hours and 20.74 hours after harvest,and this value was 7.16 hours and 4.66 hours earlier than that of wild-type melon,respectively.The value of RNAi fruits peaked at 31.81 hours and 31.65 hours after harvest,and this value was 6.54 hours and 6.25 hours later than wild-type melon in this two RNAi fruits,respectively.The respiratory peak value of CRISPR-Cas9 gene edited fruits of CmERFI-15 and CmERFII-9 occurred at 33.44 hours and 32.24 hours after harvest,and this value was 8.04 and 6.84 hours later than wild-type fruits.In addition,the fruit firmness in the CmERFI-15 and CmERFII-9 overexpression lines decreased rapidly when compared with the wild type fruits,however,the fruit firmness in the RNAi lines and gene edited lines decreased slowly comparing with the wild-type fruits.6.The expressions of ACS and ACO in transgenic fruits were quantified via qPCR,and the results showed that at the pre-climacteric stage the expression of CmACOl greatly differed between the wild-type and CmERFI-15 transgenic fruits.It was found that the expression of CmERFI-15 and CmACOl was positively correlated.At the pre-climacteric and climacteric stage,the expression level of CmAC03 were significantly different between transgenic fruits of CmERFI-15 and CmERFII-9 and the wild type.There was a positive correlation between the expression of CmERFI-15 and Cm ACO 3,and the same correlation was exhibited between CmERFII-9 and CmAC03.At the climacteric stage,the expression level of CmACS5 were significantly different between transgenic fruits of CmERFII-9 and the wild type.There was also a positive correlation between the expression of CmERFII-9 and CmACS5.Overall,the above study showed that CmEFI-15 and CmmERFII-9 accelerates melon fruit ripening.