金线莲ArFLS基因的克隆、亚细胞定位与表达分析OA

Anoectochilus roxburghii is a valuable traditional Chinese medicinal herb,primarily due to its rich content of flavonols,which are key bioactive components.However,the biosynthetic mechanism of flavonols in this species remains unclear.In this study,the flavonol synthase gene(FLS)from A.roxburghii(designated ArFLS)was cloned using full-length transcriptome sequencing data.The open reading frame(ORF)of ArFLS is 1 032 bp in length,encoding a 344-amino acid protein.Bioinformatics analysis revealed that the ArFLS protein has a molecular weight of 38.87 kDa and a theoretical isoelectric point(pI)of 5.37.It is predicted to be a stable,hydrophilic protein and contains a conserved 2-oxoglutarate-dependent dioxygenase(2-ODD)domain,characteristic of the FLS family.Phylogenetic analysis demonstrated that ArFLS shares high sequence similarity with FLS proteins from Cymbidium floribundum and Dendrobium officinale,with over 74%sequence identity.Subcellular localization assays revealed that ArFLS is distributed in both the nucleus and the cytoplasm.Expression pattern analysis indicated that ArFLS is predominantly expressed in floral tissues.Transcript levels of ArFLS in flowers were 5.95-fold higher than those in leaves,5.50-fold higher than in stems,and 2.17-fold higher than in roots,suggesting a potential role in floral development or pigmentation.Furthermore,ArFLS expression was significantly modulated by abiotic stress,with elevated expression observed under heat stress,with peak expression at 35℃,and suppressed expression under low temperatures.Prokaryotic expression and western blot analysis confirmed successful expression of ArFLS in Escherichia coli,validating its molecular properties and laying groundwork for further functional assays.Collectively,these results provide critical insights into the role of ArFLS in flavonol biosynthesis and stress responses,and establish a foundation for future studies on its regulatory mechanisms in A.roxburghii.