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Abstract：
Dendrobium sinense, an endemic medicinal herb in Hainan Island, is rich in bibenzyl compounds. However, few studies have explored the molecular mechanisms of bibenzyl biosynthesis. This study presents a comprehensive analysis of DsBBS1 and DsBBS2 function in D. sinense. A molecular docking simulation revealed high-resolution three-dimensional structural models with minor domain orientation differences. Expression analyses of DsBBS1 and DsBBS2 across various tissues indicated a consistent pattern, with the highest expression being found in the roots, implying that they play a pivotal role in bibenzyl biosynthesis. Protein expression studies identified optimal conditions for DsBBS2-HisTag expression and purification, resulting in a soluble protein with a molecular weight of approximately 45 kDa. Enzyme activity assays confirmed DsBBS2\'s capacity to synthesize resveratrol, exhibiting higher Vmax and lower Km values than DsBBS1. Functional analyses in transgenic Arabidopsis demonstrated that both DsBBS1 and DsBBS2 could complement the Atchs mutant phenotype. The total flavonoid content in the DsBBS1 and DsBBS2 transgenic lines was restored to wild-type levels, while the total bibenzyl content increased. DsBBS1 and DsBBS2 are capable of catalyzing both bibenzyl and flavonoid biosynthesis in Arabidopsis. This study provides valuable insights into the molecular mechanisms underlying the biosynthesis of bibenzyl compounds in D. sinense.
摘要：
石斛,海南岛的一种特有药材，富含二苄基化合物。然而,很少有研究探索生物合成联苄的分子机制。本研究全面分析了DsBBS1和DsBBS2在D.sinense中的功能。分子对接模拟揭示了具有微小域取向差异的高分辨率三维结构模型。DsBBS1和DsBBS2在各种组织中的表达分析显示出一致的模式，在根部发现最高的表达，这意味着它们在联苄生物合成中起着关键作用。蛋白质表达研究确定了DsBBS2-HisTag表达和纯化的最佳条件，产生分子量约为45kDa的可溶性蛋白质。酶活性测定证实了DsBBS2合成白藜芦醇的能力，表现出比DsBBS1更高的Vmax和更低的Km值。转基因拟南芥中的功能分析表明，DsBBS1和DsBBS2都可以补充Atchs突变体表型。DsBBS1和DsBBS2转基因株系的总黄酮含量恢复至野生型水平,而总的联苄含量增加。DsBBS1和DsBBS2能够催化拟南芥中的联苄和类黄酮生物合成。这项研究为D.sinense中生物合成联苄化合物的分子机制提供了有价值的见解。
