Cloning And Preliminary Function Analysis Of Homologous SPL Genes In Cucumber (Cucumis Sativus L.)

The interaction of miRNAs and their targets is broadly involved in plant development,and in a fine-tuning regulation of gene expression.The targets of miR156 are SPLs which belongs to a family of plant specific transcription factors(TFs).The interaction between them plays important roles in promoting plant development and phase change.Cucumber(Cucumis sativus.L)is one of the popular worldwide economic crops,which is also becoming a good material in plant molecular biology research.Our previous experiments verified that the expression of miR156 was lower in female buds than in male ones in cucumber.However,two of their targets,CsSPL9 and CsSPL13,showed exactly the opposite,expression of which was lower in male buds than in female ones.We believe there are similar miRNA-TFs regulation pathways in cucumber according to those facts,and these pathways are probably taking part in cucumber sex differentiation.There are no reports of genes from CsmiR156-CsSPL interaction systems,nor their roles and mechanisms in cucumber development.In the study herein,the differentially expressed CsSPL genes in cucumber male and female buds were identified and verified.We then tested their expression patterns,cloned them and constructed over-expression vectors,transformed them into Arabidopsis and analysis phenotypes of transgenic plants,with the porpose to illustrate the potential roles of CsSPLs.The main results are as follows:1.Two differentially expressed CsSPL genes in cucumber male and female buds were identified and verified.The gene of Csa002608 and Csa005294 were named CsSPL9 and CsSPL13 according to sequence similarity.2.These two genes altered obviously between different growing times,and had very different expression patterns between wild-type plants and subgynoecious plants.3.These two genes were cloned and constructed in over-expression vectors of 35S::CsSPL9 and 35S::CsSPL13.4.These vectors were transformed into Arabidopsis,and finally 7 homologous 35S::CsSPL9 and 10 homologous 35S::CsSPL13 transgenic plants were obtained.5.Ectopic expression of CsSPL9 and CsSPL13 in Arabidopsis led to elongation of primary roots and advanced bolting and flowering time.6.Ectopic expression of CsSPL9 and CsSPL13 in Arabidopsis triggered the expression of flowering-relative genes FT,API,FUL and AGL42,but decreased the level of SOC1,7.Ectopic expression of CsSPL9 and CsSPL13 in Arabidopsis showed no obvious impact on leaf development,siliques and tilling numbers,and the height of grown-up plants.Our study showed that CsSPL9 and CsSPL13 genes can influence the procedure of plant development and phase transition.They promote the development of plants probably by triggering the early accumulation of flowering genes.These results illustrated that the interaction of miR156/157-CsSPL and similar regulatory pathways were also existed in cucumber.We believe this research provides basis for further study on molecular mechanism of miR156 and SPLs in cucumber.