Shandong Provincial Key Laboratory of Agricultural Microbiology, Department of Plant Pathology, College of Plant Protection, Shandong Agricultural University, Tai'an, Shandong 271018, China.
Plant Physiol. 2021 Jun 11;186(2):853-864. doi: 10.1093/plphys/kiab146.
Cucurbits are economically important crops worldwide. The genomic data of many cucurbits are now available. However, functional analyses of cucurbit genes and noncoding RNAs have been impeded because genetic transformation is difficult for many cucurbitaceous plants. Here, we developed a set of tobacco ringspot virus (TRSV)-based vectors for gene and microRNA (miRNA) function studies in cucurbits. A TRSV-based expression vector could simultaneously express GREEN FLUORESCENT PROTEIN (GFP) and heterologous viral suppressors of RNA silencing in TRSV-infected plants, while a TRSV-based gene silencing vector could knock down endogenous genes exemplified by PHYTOENE DESATURASE (PDS) in Cucumis melo, Citrullus lanatus, Cucumis sativus, and Nicotiana benthamiana plants. We also developed a TRSV-based miRNA silencing vector to dissect the functions of endogenous miRNAs. Four representative miRNAs, namely, miR159, miR166, miR172, and miR319, from different cucurbits were inserted into the TRSV vector using a short tandem target mimic strategy and induced characteristic phenotypes in TRSV-miRNA-infected plants. This TRSV-based vector system will facilitate functional genomic studies in cucurbits.
葫芦科作物在全球范围内具有重要的经济价值。目前，许多葫芦科作物的基因组数据已经可用。然而，由于许多葫芦科植物的遗传转化困难，葫芦科基因和非编码 RNA 的功能分析受到了阻碍。在这里，我们开发了一套基于烟草环斑病毒（TRSV）的载体，用于研究葫芦科植物中的基因和 microRNA（miRNA）功能。基于 TRSV 的表达载体可以在 TRSV 感染的植物中同时表达 GFP 和异源病毒 RNA 沉默抑制剂，而基于 TRSV 的基因沉默载体可以敲低内源性基因，例如在甜瓜、西瓜、黄瓜和烟草原生质体中敲低 PDS 基因。我们还开发了基于 TRSV 的 miRNA 沉默载体，以剖析内源性 miRNA 的功能。使用短串联靶标模拟策略，将来自不同葫芦科植物的四个代表性 miRNA（miR159、miR166、miR172 和 miR319）插入到 TRSV 载体中，并在 TRSV-miRNA 感染的植物中诱导出特征性表型。该基于 TRSV 的载体系统将有助于葫芦科植物的功能基因组研究。
