Cloning and expression analysis of SsFLS2 gene in Solanum sisymbriifolium Lam.
[Objective]The study aimed to clone the FLS2 gene from wild eggplant species(Solanum sisymbriifolium Lam.)in Yunnan prov-ince,and to analyze the physicochemical properties,subcellular localization and phylogenetic evolution of its encoded protein,as well as the expression,in order to preliminarily explore the biological functions of the wild eggplant FLS2 gene under the stress of verticillium wilt.[Method]In the study,the FLS2 gene,named SsFLS2,was cloned based on the transcriptome data(S.sisymbriifolium inoculation with verti-cillium pathogens).The physical and chemical properties of SsFLS2 gene were analyzed by using bioinformatic analysis software,and real-time fluorescence quantitative PCR(RT-qPCR)was used to detect the expression of S.sisymbriifolium roots,stems and leaves,as well as the expression at different times after inoculation of verticillium pathogens.[Result]The SsFLS2 gene was 3655 bp in length,with a complete open reading frame(ORF),and encoded 1126 amino acids.The molecular formula of the encoded protein was C5596H8814N1494O1627S4,the theoretical molecular weight was 124.34 kD,the theoretical isoelectric point(pI)was 7.33,and the total mean hydrophilicity coefficient was 0.081.The secondary structure of SsFLS2 was mainly composed of α-helix(40.23％),random coil(42.81％),β-folding(3.73％),and extended chain(13.23％),and there was a transmembrane structure,localized on the cell membrane,in which the sites could be phosphoryl-ated and exceeded the threshold line,a total of 151.The results of homologous sequence alignment and phylogenetic evolutionary analysis showed that the amino acid sequence of SsFLS2 protein was most closely related to the potato(Solanum tuberosum)homologous protein(XP 006358149.2).RT-qPCR assay indicated that SsFLS2 gene was expressed in roots,stems,and leaves of S.sisymbriifolium,and the relative expression level of SsFLS2 gene was highly significant higher in roots and leaves than in stems.The relative expression level of SsFLS2 gene was substantially and highly significantly higher at 24 hours in both treatment and control groups than at other time points within 72 hours af-ter inoculation with verticillium pathogens.[Conclusion]SsFLS2 gene is successfully cloned from S.sisymbriifolium,and its expression as well as the physicochemical properties of its encoded protein are analyzed.The results showes that SsFLS2 gene is closely correlated to the re-sponse of S.sisymbriifolium to verticillium wilt stress,which lays a theoretical foundation for further study on the role of SsFLS2 gene in verti-cillium wilt resistance in S.sisymbriifolium.
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