The Research On The Mechanism Of CsPID Regulating Leaf Morphogenesis And Ovule Development In Cucumber

Cucumber(Cucumis sativus L.2n=2x=14),one of the top ten vegetables in the world,has a large demand in the vegetable market in China and has very high economic value.Cucumber,as a model crop of vegetable crops,has palm-like leaves,tendrils,unisexual flowers and parthenocarpy.Taking cucumber as the research object can contribute to studying the molecular and physiological mechanism in the morphogenesis process of above traits,and providing reliable basis for the research of the related traits.In this study,in order to further study the molecular mechanism of cucumber organ morphogenesis,the phenotypic identification and genetic analysis of cucumber round leaf mutant(rl),were carried out by screening the cucumber EMS mutant library.Gene mapping and molecular mechanisms were performed.The main results are as follows:1)There are some phenotype changes between the rl mutant and wild type: the leaf shape changes from palm to round,the number of tendrils is significantly reduced and the ovule initiation is defective,the stamens are abnormal and the petals margin is rounded,which resulting in complete female infertility and partial sterility of male organs in the rl mutant.2)The candidate gene was mapped to chromosome 1 by whole genome re-sequencing of Mut Map+method.The 22 SNPs were selected to determine the candidate gene by genotyping test,the results showed that Csa1G537400(CsPID)was co-segregated with the phenotypic data in F2 population among the 22 SNPs,the F1 generation of rl crossed with the F1 generation of the same phenotype mutant C356 for allelic test.It was further determined that CsPID regulates the formation of cucumber leaves and floral organs.The overexpression of 35S::CsPID was transformed into the Arabidopsis pid-14 mutant,and the transgenic line was obtained.The phenotype of the transgenic line rescued partial fertility compared with the pid-14 mutant,indicating that the function of CsPID is conserved in Arabidopsis and participates in the development of floral organs,and finally determined that CsPID was the casual gene for rl mutant.3)In situ hybridization results indicated that CsPID was mainly expressed in the primordia of leaves,apical meristems,petals,sepals and ovary,and the results of q RT-PCR were also consistent with the In situ hybridization.IAA immunelocalization also indicated that IAA is enriched in the placenta and ovule primordium of the wild type ovary,and there is no IAA enrichment in rl mutant.4)The subcellular localization results also showed that CsPID was localized in the cellperiphery and nucleus periphery,while the Cspid localization was different,a dot-like distribution appeared on the cell periphery,suggesting that the mutation in rl mutant might disturb the subcellular localization pattern.5)Through RNA-seq and differentially expressed gene classification of wild type and rl mutant,genes related to auxin polar transport and ovary development were selected,and CsPID might affect the development of lateral organs and ovary of cucumber by regulating the expression of these genes.6)Yeast two-hybrid results showed that CsPID could interact with the Cs REV at the protein level.The results of Bi FC further confirmed that CsPID and Cs REV co-localized in the cell periphery and nucleus periphery.