Functional Validation And Molecular Mechanism Analysis Of The Spine Base Gene CsSBS1 In Cucumber

Fruit spine is an important trait in cucumber,affecting not only commercial quality,but also fruit smoothness,transportation and storage.However,the molecular mechanism underlying the regulation of cucumber spine base remains largely unknown.We obtained a C2H2 zinc finger transcription factor CsSBS1,which controls the size of the fruit spur base,by map-based cloning using a pair of near-isogenic lines with distinct differences in fruit spine base size as parents in the previous stage.In this study,we analyzed the expression pattern and molecular mechanism of the fruit spine base size gene CsSBS1,and analyzed its differentially expressed genes using transcriptome sequencing to explore the possible hormonal pathways involved in the regulation of fruit spine base size.The results of this study not just enrich the regulatory network of cucumber fruit spur base formation,but also have important practical implications for cucumber quality breeding.The major findings are as follows:(1)Phenotypic investigations of L-SB(large spine base)and S-SB(small spine base)showed that the spine base of S-SB was significantly smaller than L-SB.Paraffin sections revealed that the smaller spine base of S-SB was due to a decrease in cell number rather than a decrease in cell volume.(2)qRT-PCR results showed that CsSBS1 was expressed in fruit spines,pericarp and ovary,with the highest expression in fruit spines and almost no expression in roots,stems,leaves and male flower buds.The detection of CsSBS1 expression in the ovary at different periods showed that CsSBS1 expression was highest in the-2DPP ovary,after which CsSBS1 expression gradually decreased.GUS staining and in situ hybridization assays showed that CsSBS1 was expressed in high abundance in the fruit spines and near the pericarp.These results suggest that CsSBS1 is specifically expressed in fruit spines.(3)Overexpression of CsSBS1 in S-SB resulted in significantly larger spine base in the overexpression strain compared with the control;introduction of CsSBS1-RNAivector in L-SB resulted in significantly smaller spine base in the RNAi strain compared with L-SB,indicating that CsSBS1 positively regulates fruit spine base size.Overexpression of CsSBS1 in wild-type Arabidopsis resulted in a significant increase in the number of Arabidopsis stem,leaf and inflorescence trichome,and more stem and leaf trichome branching,indicating that CsSBS1 has different roles in Arabidopsis trichome development.(4)Using the CsSBS1-AD decoy vector,the fruit spine development gene Cs TTG1 was screened by a yeast library,and yeast two-hybrid(Y2H)analysis showed that Cs TTG1 and CsSBS1 directly interacted with each other in vitro.In addition,bimolecular fluorescence complementation(Bi FC)analysis and fluorogenic enzyme complementation(LCI)analysis similarly confirmed this result.The complete absence of CsSBS1 in the small base material S-SB and the overexpression of Cs TTG1 in S-SB resulted in larger fruit spine base,suggesting that CsSBS1 is located upstream of Cs TTG1 regulating fruit spine base development.(5)Yeast triple hybridization(Y3H)analysis showed that the trichome initiation development gene Cs GL1 can interact indirectly through Cs TTG1 and CsSBS1.Similarly,bimolecular fluorescence complementation(Bi FC)analysis,fluorophore complementation(LCI)analysis and immunoprecipitation(Co-IP)analysis verified this result,indicating that CsSBS1/Cs TTG1/Cs GL1 co-regulate cucumber fruit spine base development as a trimeric complex.(6)The L-SB and RNAi strains were subjected to transcriptome sequencing to screen for differentially expressed genes,and it was found that ethylene-related differentially expressed genes were much higher than other phytohormones,and q RT-PCR showed that only ethylene-related genes were significantly different,and endogenous hormone measurements similary showed that only ethylene content was significantly different.