| Rapid fruit swelling is a typical characteristic of fruit development in gourd fruit vegetable crops.The increase of cucumber fruit volume is mainly driven by the fruit cell division and enlargement.Previously,our research group obtained a short fruit mutant Csvfb1 from the cucumber EMS mutagenic mutant library,and used the Mut Map method to locate the mutant gene and named it CsVFB1.CsVFB1 encodes the F-box protein,which may involve in the regulation of cucumber fruit development particularly through ubiquitination and degradation of the substrate.Therefore,finding substrate protein is an important step to reveal the regulatory mechanism of the CsVFB1 gene.By comparing the differences in plant type,morphological and histocytological differences in fruit development between cucumber variety 406 and mutant Csvfb1,the quantitative technology of protein DIA,and screening and verifying direct ubiquitination substrates,this study was conducted to reveal the molecular mechanism and role of CsVFB1 in fruit development regulation.The following results were obtained:1.Mutant plant have shown significantly shorter plant height,curled leaves,narrowed leaf margins,thicker stems,and inter-node changes compared with wild-control plants.2.Morphological indicators such as transverse and longitudinal diameter,number of ventricles s after flowering at different days,and histocytological indicators such as mesocarp cell area and cell number between wild control and Csvfb1 it was observed that the CsVFB1 mutation resulted in an increase in the number of ventricles,and in the proportion of four-ventricle fruits,which led to an increase in the number of cross-sectional cells.However,expansion of the cross-sectional cells were inhibited,which cause thicker transverse diameter in the mutant fruit phenotype..CsVFB1 mutation Suppressed longitudinal section cell enlargement which shorten the longitudinal diameter of the fruit3.In order to explore how CsVFB1 mutation affects cell expansion,it is important to screen its ubiquitinated substrates.CsVFB1 mutation inhibits the ubiquitination of the substrate by the SCF complex and affects its degradation through26 S proteasome.MG132 is a ubiquitinated protease inhibitor which inhibit the activity of the 26 S proteasome,thereby affecting all ubiquitin proteasome pathway.Therefore,both CsVFB1 mutation and MG132 treatment would cause to a large accumulation of CsVFB1 substrate proteins.In current study,,we used the protein DIA quantitative technology and screened significantly upregulated protein in the peel of the Csvfb1 mutant and significantly accumulated in the WT after treatment with MG132 as the preliminary candidate substrate.A total of 19 candidate substrates were screened..and further experiments such as firefly luciferase complementation and yeast two-hybrid experiments were conducted to identify the direct interaction between CsVFB1 protein and candidate substrates.These results showed that FE1 and FE8 proteins have direct interactions with CsVFB1,which may be the ubiquitinated substrates of CsVFB1 protein. |
