Abstract

Pepper (Capsicum annuum L.) is one of the most widely cultivated species and is highly valued for its pungency. The pungency of pepper is primarily attributed to a group of chemical compounds known as capsaicinoids. These compounds are synthesized in the placental tissue of pepper fruits through the activation of specific genes and enzymes, thereby contributing to their pungency. Protoplast-based gene expression systems have been considered an efficient method for gene function studies, protein-protein interactions, promoter analysis, and subcellular localization. Here, we optimized an efficient protocol for isolating protoplasts from pepper placental tissues and the polyethylene glycol (PEG)-mediated transient expression of green fluorescent proteins (GFP). Several factors affecting GFP expression in intact protoplasts were evaluated and optimized in this study. Protoplast isolation was carried out using 2.0% cellulase “onozuka” R-10 and 0.3% macerozyme R-10 solution. Different amounts of plasmid DNA and various incubation times for transfection with 40% PEG resulted in different transfection efficiencies (78–85%). The highest GFP transformation efficiency was observed when 120 µL protoplast suspension (3 × 10⁷ to 5 × 10⁷/mL) was mixed with 15 µg plasmid DNA and incubated for 25 min with an equal volume of 40% PEG. The improved protocol described in this study can be helpful for the isolation and transfection of pepper placenta-originated protoplasts and for the rapid investigation of pepper gene functions.

中文翻译：

---

辣椒（Capsicum annuum L.）胎盘来源原生质体分离和瞬时表达的优化方案

摘要

胡椒 ( Capsicum annuum L.) 是种植最广泛的物种之一，因其辛辣而受到高度重视。胡椒的辛辣味主要归因于一组称为辣椒素的化合物。这些化合物是通过激活特定基因和酶在辣椒果实的胎盘组织中合成的，从而增强了辣椒的辛辣味。基于原生质体的基因表达系统被认为是基因功能研究、蛋白质-蛋白质相互作用、启动子分析和亚细胞定位的有效方法。在这里，我们优化了一种有效的方案，用于从辣椒胎盘组织中分离原生质体和聚乙二醇（PEG）介导的绿色荧光蛋白（GFP）的瞬时表达。本研究评估并优化了影响完整原生质体中 GFP 表达的几个因素。使用2.0%纤维素酶“onozuka”R-10和0.3%macerozyme R-10溶液进行原生质体分离。不同量的质粒 DNA 和不同的 40% PEG 转染孵育时间会导致不同的转染效率 (78–85%)。^{当 120 µL 原生质体悬浮液（3 × 10 7}至 5 × 10 ⁷ /mL）与 15 µg 质粒 DNA 混合并与等体积的 40% PEG 一起孵育 25 分钟时，观察到最高的 GFP 转化效率。本研究中描述的改进方案有助于辣椒胎盘来源的原生质体的分离和转染以及辣椒基因功能的快速研究。