Cloning and Subcellular Localization Analysis of GGPPS in Bletilla striata
WANG Shuang-Shuang1, XIAO Xin-Yuan1, DOU Quan-Li1, YUAN Xin-Yue1, LIU Ting1, XU Zhi-Wen2,*
1 College of Biological and Agricultural Science and Technology, Zunyi Normal University, Zunyi 563006, China; 2 Forestry Bureau of Zunyi City, Zunyi 563000, China
Abstract：Terpenoids serve as the fundamental material basis for the medicinal value of Bletilla striata. Geranylgeranyl pyrophosphate synthase (GGPPS) is a key branch point enzyme in the terpenoid metabolic network. However, the characteristics of the BsGGPPS gene remain unclear. In this study, using the wild purple-flowered B. striata as material, the full-length CDS of the BsGGPPS gene was cloned via RT-PCR based on transcriptomic data. Bioinformatics tools were employed to analyze the physicochemical properties, functional domains, and evolutionary relationships of its encoded protein. A fusion expression vector was constructed and transiently transformed into tobacco (Nicotiana tabacum) to determine the subcellular localization of the BsGGPPS protein. Furthermore, its expression levels across different tissues of B. striata were assessed using qRT-PCR. The results showed that the cloned BsGGPPS CDS was 1 083 bp in length, encoding 360 amino acids. Bioinformatics analysis revealed that BsGGPPS was a weakly hydrophobic, unstable protein without a signal peptide or transmembrane domains. It contained conserved aspartate-rich catalytic motifs (FARM and SARM) and belonged to a significant clade within the isoprenyl pyrophosphate synthase family. Subcellular localization indicated that BsGGPPS was primarily distributed in the cytoplasm and plastids. Phylogenetic analysis demonstrated that BsGGPPS formed a monophyletic group with GGPPS from Dendrobium catenatum and Phalaenopsis equestris. qRT-PCR results revealed that the expression level of BsGGPPS in the pseudobulbs of B. striata seedlings was significantly higher than that in the roots, stems, and leaves. These findings suggested that BsGGPPS was likely involved in the terpenoid biosynthesis pathway of B. striata through coordinated action in plastids and the cytoplasm. Its tissue-specific high expression in pseudobulbs implied a putative regulatory role in the storage organs for medicinal terpenoid active compounds. This study not only provides a theoretical foundation for elucidating the regulatory mechanisms of terpenoid metabolism in B. striata but also lays the groundwork for subsequent genetic engineering approaches aimed at directional regulation of the biosynthesis of medicinal terpenoids in B. striata.
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