Cloning，expressional characterization and subcellular localization of TkGASA1 gene in rubber dandelion （Taraxacum kok-saghyz Rodin）

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Objective Rubber dandelion （Taraxacum kok-saghyz Rodin，TKS） is one of the most promising alternative rubber-producing plants.A better comprehension of the regulation mechanisms of its growth and reproductive development is essential for its genetic improvement.The cysteine-rich small peptide gene family，Gibberellic acid-stimulated Arabidopsis （GASA），plays critical roles in various plant growth and reproductive processes.Cloning the GASA gene from TKS and analyzing its expression patterns and subcellular localization can provide valuable insights into the functional roles of the GASA genes in the growth and reproductive development of TKS. Method Using the TKS germplasm line TK20 as the experimental material，a GASA gene with high expression in flowers was screened from transcriptome data and named TkGASA1.The TkGASA1 gene was cloned and subjected to bioinformatics analysis.Tissue-specific and floral organ-specific expression patterns of TkGASA1 were detected using real-time quantitative PCR （qPCR）.Additionally，the subcellular localization of TkGASA1 was examined through transient transfection assays in tobacco. Result The coding sequence （CDS） of TkGASA1 was successfully cloned，with a total length of 309 bp.The encoded TkGASA1 protein contains an N-terminal signal peptide，a highly variable hydrophilic region in the middle，and a C-terminal GASA domain rich in 12 cysteine residues.TkGASA1 is a hydrophilic protein，with its secondary structure composed of random coils and alpha helices.The tertiary structure of TkGASA1 shows high similarity to the AaGAST1 protein from Artemisia annua.TkGASA1 was highly expressed in flowers，weakly expressed in pedicels and leaves，and almost undetectable in roots and latex.Among different floral organs，TkGASA1 exhibited higher expression in styles，stigmas，pappus，and beaks.Subcellular localization results indicated that the TkGASA1-GFP fusion protein was localized in both the cytoplasm and nucleus. Conclusion The TkGASA1 gene from TKS encodes a 102-amino acid protein with typical GASA structural features.The TkGASA1 protein is localized in the cytoplasm and nucleus.TkGASA1 is highly expressed in flowers，particularly in styles，stigmas，pappus，and beaks.This study lays a theoretical foundation for further functional characterization of the TkGASA1 gene.