Molecular Mechanism Of Regulation Of The Floral Sex Types In Cucumis

The flower development is the basis of fruit and seeds in plants.Differential development of pistils and stamens in a given flower forms unisexual female and male flowers,thus forming floral sex type diversity.Moreover,the various distribution of flowers with different sex types on a same plant forms plant sexual diversity.Therefore,it is an important part of plant developmental biology and agricultural production to study the regulation mechanism of flower and plant sexual types.Cucumis species includes melon and cucumber,both of which are main cultivated vegetables.The two species have abundant floral and plant sexual types,and the regulation of sexual differentiation can directly affect their yield and quality.This study focuses on the regulation of sex-controlling genes in melon and cucumber.Identifying the sex type mutants in melon and cucumber helps to discover new genetic resources.Identifying the sex-related ethylene signal factors helps to confirm the interaction between ethylene and sex-controlling genes.Genomic SSR markers and linkage analysis helps to fine-mapping two novel melon sex-related genes.At the end of this study,a gene interaction model is conducted to explain the sex expression in melon and cucumber.The detailed results are following:1.Identification of genetic background of existing melon and cucumber mutant resources.Using gene sequencing,gene expression and genetic analysis,the sex-genotype of cultivated melon materials 0426,0544 and 998 are identified,and a new female enhancement gene In-F and a recessive hermaphroditic gene h are identified in a semi-wild strain Y101.In addition,three cucumber F gene(Female)mutants with the same genetic background are identified by genome re-sequencing and gene expression analysis.2.Verification of the ethylene sensitivity in the expression of sex-controlling genes.Various ethylene related treatments are applied to melon and cucumber mutants.The results reveal that,the expression of Cs ACS1,Cs ACS2,Cs ACS11,Cm ACS7 and Cm ACS11 are ethylene-induced,and the inducibility is sensitive to measurement and time of ethylene treatment.Transformation studies show that the promoter regions of Cs ACS11 and Cm ACS11 genes are also sensitive to ethylene in tobacco plants.3.Identification of the sex-related ethylene signal components.Five ethylene signaling components are identified by RNA-Seq using the cucumber F mutants,and the expression of their coding genes are verified to be ethylene responsive.Then ethylene response factor(ERF)is analyzed,and cucumber Cs ERF110 and melon Cm ERF110 are cloned.Sequence analysis shows that both of them have typical ERF characteristics.Cs ERF110 has ethyleneand sex-specific expression pattern,and its protein is nuclear localized,indicating that it may participate in ethylene signal transduction.4.Identification of the interaction between ERF110 and ACS11 gene.It is predicted that the promoter region of Cs ACS11 gene has potential binding sites of ERF.Using Yeast one-hybrid system and tobacco heterologous Ch IP-PCR analysis,it is found that there might be two Cs ERF110 binding sites in the promoter,and one site has non-conserved cis-acting element sequence.Expression activity analysis show that Cs ERF110 and Cm ERF110 can induce the expression activity of CsACS11 and CmACS11 promoters,respectively.These results indicate that ethylene can activate the expression of ACS11 via ERF110 in melon and cucumber.5.Fine-mapping of the In-F and h loci in melon.Crossing and genomic SSR analysis reveal that Y101 is conservative in melon genome sequence,but the large number of SSR polymorphisms indicates that Y101 belongs to melon related species.Genetic analysis further confirms that In-F is a dominant single gene,and h is different from m gene.After fine-mapping analysis,the In-F locus is located between SSR markers Cm SSR10023 and Cm SSR10034,which includes a 62.3 kb region in melon genome Chr03.Gene annotation indicates that there are 7 coding genes,and Cm ACS11(MELO3C010779)is probably the dominant gene controlling enhanced female phenotype in Y101.In addition,fine-mapping analysis located the h locus between SSR markers Cm SSR20029 and Cm SSR20034,which includes a 37.4 kb interval in melon genome Chr08.Gene annotation reveals that there are8 coding genes,including a gene cluster containing 7 cytochrome P450 genes and one receptor protein kinase gene.Based on the current results and previous knowledge,an ethylene-based molecular model in the melon and cucumber sex-controlling genes interaction is proposed.It is concluded that ethylene is not only the product of sex-controlling genes,but also the induced substance for these gene expression,which needs to be achieved through ERF.Different induced expression of these genes can produce different levels of ethylene,and induce pistil and arrest stamen development,respectively,then resulting in changes in flowers and plant sex types.