Transcriptional Regulation Mechanism About Melon Bisexual Flower Emerged Under Ethephon Treatment

Melon(Cucumis melo L)is an important horticultural and economic crop that widely cultivated in the worldwide which flower buds can differentiate to different sex types,and composition to abundant types of sex differentiation.In the normal production,most of the melons are andromonoecious in which male flowers are arose both in main stem and lateral branches but but bisexual flowers only arose from the leaf axils of lateral branches,and the fruits are developed from bisexual flowers.This study is mainly to search the molecular mechanism of the production of bisexual flowers in the andromonoecious melon plants.In this study,RNA-seq was applied on two comparison sets:shoot apex of lateral branches(LA)与 shoot apex of main stem(MA),and shoot apex of main stem after ethephon treatment(Eth)与control(Cont).The RNA-seq data was sorted and analyzed that have found some of the key KEGG pathways related to the production of bisexual flowers and the mechanism of the significant difference genes in the key pathways was analyzed.The major discoveries are:1.Bisexual buds emerged in the leaf axil of main stem after the application of ethephon.Compared with the O number of bisexual flowers produced by the control within 20 nodes of the main stem of the treated plant of average,2.26 bisexual flowers were produced,and the bisexual flowers can develop into fruits.These results suggested that bisexual flowers were normally produced in lateral branches and the application of ethephon can effectively induce the formation of bisexual flowers in main stem.2.Twelve libraries were constructed through high-throughput RNA-seq sequencing,and a total of 664 million Raw reads were obtained.Among them,in the transcription factor data of MA vs LA,2060 genes were up-regulated and 1,944 genes were down-regulated.,A total of 4,004 significantly differentially expressed genes(DEGs).In the Eth vs Cont transcription factor data,a total of 2839 significantly differentially expressed genes were found,of which 1,742 were up-regulated genes and 1,097 were down-regulated genes.And in the two pairs of comparative samples,a total of 631 up-regulated differential genes and 492 down-regulated differential genes were found to be co-expressed..3.The KEGG pathway obtained from the enrichment analysis of the significantly different genes shows that the DEGs that are jointly up-regulated in the two samples of LA 与 MA and Eth 与 Cont are termed "plant hormone signal transduction","MAPK signal transduction pathway" and "carbon metabolism" in the KEGG term.Significantly enriched in the three pathways,and analyzed the two sets of samples from LA 与 MA and Eth与 Cont to upregulate the expression of genes.Among them,there are 6 transcription factors involved in the ethylene signaling pathway.The analysis is presumed to be related to CmETR1,CmEBFs,CmEIN3,CmEILl and CmERF1,Which are involved in encoding AUX1(MELO3C003035),AUX/IAA(MELO3C003473)and SAURs(MELO3C006771 and MELO3C022337)in transcription factors related to the auxin signaling pathway,and 5 genes related to the MAPK cascade signaling pathway Transcription factors are involved in coding:WRKY transcription factors(MELO3C020489 and MELO3C009127),mitogen-activated protein kinase(MELO3C020718)and catalase(MELO3C017023 and MELO3C026532),and there are 20 differential genes that are co-expressed up-regulated in the "carbon metabolism" pathway4.Bioinformatics and expression analysis of different parts of the co-expressed gene CmERF1 with the most significant difference in ethylene signaling pathway were carried out.The test results showed that CmERF1 and AtERF1 are closely related in Arabidopsis,and the gene structure analysis showed that CmERFl is related to AtERF1.AtERF1 has a similar exon.The structural analysis with other species showed that the AP2/ERF domain of CmERFl is similar to the AP2/ERF domain of AtERF1(89.66%),CsERF1(98.28%)and ClaERF1(100%).Subcellular analysis of CmERFl is located in the nucleus,and the expression level of CmERFl in hermaphrodite flowers and lateral vine apex is higher than that in male flower buds by fluorescence quantitative analysis,indicating that CmERF1 is involved in the expression of hermaphrodite flowers.5.Similar to most AP2/ERF family genes,CmERF1 works by combining with the GCC(AGCCGCC)box or CRT/DRE(A/GCCGAC)cis-elements in the promoter region of the downstream gene,so it can enrich the two sets of samples.The collected terms are "plant hormone signal transduction","MAPK signal pathway","carbon metabolism" and "plant-pathogen interaction" in the four KEGG pathways which are up-regulated differential genes.They were screened through editing scripts,and 15 were obtained.Predict target genes.These target genes involve brassinolide,jasmonic acid,cytokinin and other plant hormone signaling pathways,as well as phytopathogen interactions,carbon metabolism pathways,and linalin biosynthesis pathways.6.Quantitative Real-time PCR was used to analyze the expression levels of the 15 downstream target genes screened at different treatment concentrations at the same sampling time and at the same treatment concentration at different sampling times.In the experiment at the same concentration and different sampling time,we found that these genes with gene IDs MELO3C004498,MELO3C017023,MELO3C018836,MELO3C017130,MELO3C020489 and MELO3C009127 and CmERF1 treated with the same concentration of ethephon,showed similarity with the increase of treatment time.These genes are mainly involved in carbon metabolism pathways,plant pathogen interactions,and camalexin synthesis;among them,linaline biosynthesis is involved.Among them,which is related to camalexin synthesis encoding WRKYs gene ID was MELO3C009127 and the transcription factor involved in the pathogen interaction pathway,the transcription factor with gene ID MELO3C017130 showed similar expression trends with CmERF1 under these two different conditions.