Walnut (Juglans regia), an important contributor to oil production among woody plants, encounters research constraints due to difficulties in the subcellular localization and functional analysis of its proteins. These limitations arise from the protracted fruiting cycle and the absence of a reliable transient gene transformation system and organelle markers. In this study, we established a transient expression system using walnut protoplasts and generated fluorescent-tagged organelle markers, whose localization was validated against Arabidopsis (Arabidopsis thaliana) organelle markers. The versatility of this system was demonstrated through pharmaceutical treatments, confirming its ability to determine the subcellular localization of endogenous proteins. We determined the subcellular localization of walnut oleosin proteins and explored protein-protein interactions through bimolecular fluorescence complementation (BiFC) analysis. We also explored the effects of abscisic acid (ABA) signaling on oil body morphology and the regulation of walnut WRINKLED1 (JrWRI1) in lipid biosynthesis. Overall, this stable and versatile protoplast-based transient expression system, integrated with walnut organelle markers, enhances the subcellular localization and functional studies of uncharacterized walnut proteins. This advancement accelerates research into walnut gene function and streamlines molecular breeding processes with high throughput efficiency.

中文翻译：

---

原生质体瞬时转化促进核桃蛋白的亚细胞定位和功能分析


核桃 （Juglans regia） 是木本植物产油的重要贡献者，由于其蛋白质的亚细胞定位和功能分析困难，因此遇到了研究限制。这些限制源于漫长的子实体周期以及缺乏可靠的瞬时基因转化系统和细胞器标记物。在这项研究中，我们使用核桃原生质体建立了一个瞬时表达系统，并生成了荧光标记的细胞器标志物，其定位针对拟南芥 （Arabidopsis thaliana） 细胞器标志物进行了验证。该系统的多功能性通过药物治疗得到证明，证实了其确定内源性蛋白质亚细胞定位的能力。我们确定了核桃油酸蛋白的亚细胞定位，并通过双分子荧光互补 （BiFC） 分析探索了蛋白质-蛋白质相互作用。我们还探讨了脱落酸 （ABA） 信号对油脂体形态和脂质生物合成中核桃WRINKLED1 （JrWRI1） 的调节作用。总体而言，这种稳定且多功能的基于原生质体的瞬时表达系统与核桃细胞器标志物相结合，增强了未表征核桃蛋白的亚细胞定位和功能研究。这一进步加速了核桃基因功能的研究，并以高通量效率简化了分子育种过程。