Cloning And Functional Analysis Of AGL15-Like Gene In Cucumber

MADS-box gene is a family of transcription factors and play vital roles in plant growth and development.AGAMOUS-like15(AGL15-like)is a subfamily member of the MADS-box gene family,which plays an especially obvious role in plant somatic embryogenesis,floral organ transformation,leaf and fruit development.However,the AGL15-like genes in cucumber(Cucumis sativus L.)has not been reported,cloning and functional analysis of cucumber AGL15-like gene may help to clarify the molecular mechanism of floral organ development in cucumber.In this study,Arabidopsis thaliana AtAGL15 and AtAGL15 ortholog,CsMADS25 and CsMADS26,were cloned from cucumber,and the bioinformatics and expression characteristics were studied.In addition,the functions of CsMADS25 and CsMADS26 genes were verified by constructing the over-expression vectors of Arabidopsis thaliana and analysis of the transgenic Arabidopsis thaliana phenotypes.The main results are as follows:(1)The open reading frame(ORF)of CsMADS25 was 810 bp in length and encoded a deduced protein consisting of 269 amino acids.The open reading frame(ORF)of CsMADS26 was 669 bp in length and encoded a deduced protein consisting of 222 amino acids.CsMADS25 and CsMADS26 are hydrophilic acidic proteins,and both contain MADS-MEF2-like and K-box functional sites unique to MADS-box transcription factor,as well as multiple DNA binding sites.Phylogenetic tree analysis indicated that CsMADS25 was clustered into the AGL18 clade of AGL15 subfamily,then CsMADS26 was clustered with AGL18 proteins.(2)Expression analysis based on qRT-PCR showed that CsMADS25 is mainly expressed in roots and leaves,CsMADS26 is mainly expressed in flowers.Stress expression analysis showed that under the same cold,salt(NaCl),drought(PEG)and ABA treatments,The stress response of CsMADS26 gene was significantly higher than that of CsMADS25 gene,and the expression patterns of them were obviously different.(3)CsMADS25 and CsMADS26 genes were genetic transformation in Arabidopsis thaliana.The results showed that Overexpression of CsMADS25 resulted in dwarf and small-leaf phenotypes in transgenic Arabidopsis plants,transgenic Arabidopsis plants with ectopic expression of CsMADS26 exhibited curled rosette and cauline leaves,and the leaf size was much smaller than that of wild-type(WT)plants,the leaf index value(leaf length/width ratio)of transgenic plants was dramatically increased compared with that of wild-type(WT)plants.These findings suggest that CsMADS25 and CsMADS26 genes play an important role in the regulation of cucumber leaf development and provide clues for the functionalcharacterization in the future.