Study On The Function Of CsWOX1 In Regulating Petal Development And Screening Of Its Interacting Proteins

Earlier studies found that the early termination of cucumber CsWOX1 gene causes abnormal petal development,which is manifested as petals splitting and narrowing.In order to further explore the proteins that co-regulate petal development and petal development with CsWOX1,this study used yeast two-hybrid technology to screen from the cucumber apical bud c DNA library,and analyzed the AP2/ERF family member Csa6G491020(CsAP2)among the candidate proteins.The main research results are as follows:1.Compared with the cucumber AM218 varieties,the petals of the mutants with CsWOX1 gene termination prematurely narrowed,indicating that the short cut of CsWOX1 protein affected the development of cucumber flower organs.Four Arabidopsis wox1 wox3 double mutant lines were obtained by CRISPR-Cas9 technology,and the CsWOX1 gene was overexpressed in wox1 wox3 double mutant lines.By observing the petal development of Arabidopsis positive plants,it was found that overexpression of the CsWOX1 gene in wox1 wox3 double mutant lines could compensate for the phenotypic defect of petal narrowing to a certain extent,confirming that the function of CsWOX1 in the lateral expansion of petals is relatively conservative.2.The pGBKT7-CsWOX1 bait vector was constructed and verified that the bait vector did not inhibit or poison the growth of yeast and had no self-activation effect on the reporter gene.17 candidate proteins that interact with CsWOX1 transcription factor were screened from the cucumber terminal bud c DNA library.After analyzing the selected proteins,Csa6G491020(CsAP2),which affects the formation of flowers,was selected for further study,and its reliability was verified by a rotation test.The Bi FC technology was used to determine that the interaction between CsWOX1 protein and CsAP2 occurred in the nucleus.3.Gene expression analysis showed that the CsAP2 gene was expressed in all tissues of cucumber,and the subcellular localization of the protein indicated that the CsAP2 protein was distributed in the nucleus.Bioinformatics comparison found that CsAP2 is similar to At AP2.Overexpression of CsAP2 in Arabidopsis influenced the development of floral organs and down-regulated the expressions of At AG,At WUS and At ARF3,which were the member in the regulatory pathway of floral organ stem cells in Arabidopsis.The expression of At LFY,which was related to inflorescence transformation,was up-regulated in overexpression plants.