Characteristics Analysis And Gene Functional Research Of The “mango” Mutant In Cucumber (Cucumis Sativus L.)

Owing to its diversity in sex type, cucumber has become a model in the study of sex-determination in plant. It is been a long time for the research of floral organ development, while digging the key regulation factors are still the main content in current research. Cucumber "mango" mutant, with its abnormal male and female flowers, is an important material for study of the floral organ development and regulation. The main results are following:1.The morphological observation of mutant and wild-type plants showed that, in the "mango" mutant, the cotyledons were not smooth; the first and second true leaves were wedge-shaped and not flattened, and the the main veinswere elongatedfrom the third true leaves; petals were narrow in male and female flowers; the anther fail to produce pollen,and then sterile; the mutant pistilswere small and slim, and the stigma merged together partly. The pistils can not be pollinated artificially in greenhouse. However, they can be pollinated in an open environment,and then producingless seeds and mutant mango-shaped fruits.Therefore, we named it "mango" mutant. The observation of leaves, male and female flowers in different development stages found that: palisade and vascular tissue of the mutant leaves were irregularly arranged, and the number and size of the cells were affected;the development of the anther in male flowers and the pollen were inhibited; the ovule development and ovary enlargement were restricted in female flower development.2. Theprevious work had finished the mapping cloning,and the candidate gene was mapped in a 16.7kb interval in the long arm of Chromosome I, and only one candidate gene was found in this region, which is Csa1M042780, encoding a WUSCHEL-related homeobox1 protein. Because the Csa1M042780 gene is homologous to the AtWOX1 gene in Arabidopsis, we then named it CsWOX1. Further analysis indicated that the gene belongs tothe WOX transcription factor family.We also identified that the mutant phenotype was derived from a 1-bp deletion in the coding region of the CsWOX1 gene.The deletion produced a truncated protein, which lost the conserved WUS-box motif. The expression of CsWOX1/Cswox1 gene were detected in the tissues of wild type and mutant materials, such as apical bud, female flower, male flower, leaf and root, and the expression of Cswox1 gene was significantly increased in mutant.We can speculate that the change of protein structure or the increased expression of CsWOX1 may be the reasons of the mutant.3. In order to analyze thereasons of the mutant, we transferred CsWOX1/Cswox1 into the Arabidopsis plants under 35 S over-expression promoters, respectively. The phenotypic analysis of the positive transgenic Arabidopsis plants showed that overexpression of the CsWOX1 and Cswox1 had similar negative effects on the Arabidopsis floral organs and plant fertility, indicating that there were no difference in CsWOX1 and Cswox1 when they were overexpressed. The results indicated the expression regulation of this gene, rather than the protein structure mutation, should be the key-point of the functional change in mutant gene, even though the truncated protein had losed the WUS-box motif.4. The results of RNA-seq analysis in the the apical budsof mutant and wild-type materials showed that there were only the Cswox1 and CsWUS genes were involved in the WOX transcription factor family, indicating that CsWUS and Cswox1 might be important for the development regulation of male flowers.Duingto the WUS-related genes and regulatory mechanisms in Arabidopsis, we further analyzed the RNA-Seq datas and found that there were many genes involving in the flower development, just like CsWUS, CsSPL/CsNZZ,CsMYB33, CsDYT1, CsAMS, CsMYB103, CsMS1, all of which were similar to the WUS-relatedpathway genes in Arabidopsis. The expression of CsWOX1, CsWUS, CsAG,CsSPL/CsNZZ in cucumber ovary were also analyzed and found that CsWUS, CsAG,CsSPL/CsNZZwere down-regulated following the up-regulated expression of Cswox1,which in accordance with the expression patterns in male flowers. Based on the results, we found that WUS gene was involved in the similar regulating pathways with Arabidopsis,while there was no definite regulation relationship with AtWOX1 and AtWUS. RNA-seq analysis found that Cswox1 can inhibit the expression of CsWUS.Based on the results, the generation of “mango” mutantmay be closely related to the decreased expression of CsWUSand the other relative genes whichwere inhibited by the up-regulation of Cswox1.