BioAgriTech Research Group (BioATR), Faculty of Science and Natural Resources, Universiti Malaysia Sabah, Jalan UMS, 88400, Kota Kinabalu, Sabah, Malaysia.
Biotechnology Research Institute, Universiti Malaysia Sabah, Jalan UMS, 88400, Kota Kinabalu, Sabah, Malaysia.
Mol Biol Rep. 2024 Sep 20;51(1):1000. doi: 10.1007/s11033-024-09943-2.
Phalaenopsis bellina, an orchid native to Borneo, is renowned for its unique appearance. It releases distinct fragrances, which have been linked to the presence of terpenoids. However, the identification and study of sesquiterpene synthase in P. bellina remain limited. In this study, we examines the functional characterisation of terpene synthase (TPS) from P. bellina, known as PbTS, through recombinant protein expression and its manifestation in the flower.
Gene annotation of PbTS revealed that the inferred peptide sequence of PbTS comprises 1,680 bp nucleotides encoding 559 amino acids with an estimated molecular mass of 65.2 kDa and a pI value of 5.4. A similarity search against GenBank showed that PbTS shares similarities with the previously published partial sequence of P. bellina (ABW98504.1) and Phalaenopsis equestris (XP_020597359.1 and ABW98503.1). Intriguingly, the phylogenetic analysis places the PbTS gene within the TPS-a group. In silico analysis of PbTS demonstrated stable interactions with farnesyl pyrophosphate (FPP), geranyl pyrophosphate (GPP), and geranylgeranyl pyrophosphate (GGPP). To verify this activity, an in vitro enzyme assay was performed on the PbTS recombinant protein, which successfully converted FPP, GPP, and GGPP into acyclic sesquiterpene β-farnesene, yielding approximately 0.03 mg/L. Expressional analysis revealed that the PbTS transcript was highly expressed in P. bellina, but its level did not correlate with β-farnesene levels across various flowering time points and stages.
The insights gained from this study will enhance the understanding of terpenoid production in P. bellina and aid in the discovery of novel fragrance-related genes in other orchid species.
原产于婆罗洲的蝴蝶兰因其独特的外观而闻名。它会释放出独特的香气，而这些香气与萜烯类物质的存在有关。然而，对蝴蝶兰中倍半萜合酶（TPS）的鉴定和研究仍然有限。在这项研究中，我们通过重组蛋白表达及其在花中的表现来研究蝴蝶兰中的萜烯合酶（TPS），并将其命名为 PbTS，以研究其功能特征。
PbTS 的基因注释表明，PbTS 的推断肽序列由 1680bp 核苷酸编码，编码 559 个氨基酸，估计分子量为 65.2kDa，等电点为 5.4。与 GenBank 的相似性搜索表明，PbTS 与之前发表的蝴蝶兰部分序列（ABW98504.1）和 Phalaenopsis equestris（XP_020597359.1 和 ABW98503.1）具有相似性。有趣的是，系统发育分析将 PbTS 基因置于 TPS-a 组内。PbTS 的计算机分析表明其与法呢基焦磷酸（FPP）、香叶基焦磷酸（GPP）和香叶基二磷酸（GGPP）具有稳定的相互作用。为了验证这一活性，对 PbTS 重组蛋白进行了体外酶活性测定，该蛋白成功地将 FPP、GPP 和 GGPP 转化为无环倍半萜 β-法尼烯，生成约 0.03mg/L。表达分析表明，PbTS 转录本在蝴蝶兰中高度表达，但在不同开花时间点和阶段，其水平与 β-法尼烯水平无关。
本研究的结果将提高对蝴蝶兰萜烯类物质产生的认识，并有助于在其他兰花物种中发现与香气相关的新基因。
