Papaya (Carica papaya L.) is regarded as an excellent model for genomic studies of tropical trees because of its short generation time and its small genome that has been sequenced. However, functional genomic studies in papaya depend on laborious genetic transformations because no rapid tools exist for this species. Here, we developed a highly efficient virus-induced gene silencing (VIGS) vector for use in papaya by modifying an artificially attenuated infectious clone of papaya leaf distortion mosaic virus (PLDMV; genus: Potyvirus), PLDMV-E, into a stable Nimble Cloning (NC)-based PLDMV vector, pPLDMV-NC, in Escherichia coli. The target fragments for gene silencing can easily be cloned into pPLDMV-NC without multiple digestion and ligation steps. Using this PLDMV VIGS system, we silenced and characterized five endogenous genes in papaya, including two common VIGS marker genes, namely, phytoene desaturase, Mg-chelatase H subunit, putative GIBBERELLIN (GA)-INSENSITIVE DWARF1A and 1B encoding GA receptors; and the cytochrome P450 gene CYP83B1, which encodes a key enzyme involved in benzylglucosinolate biosynthesis. The results demonstrate that our newly developed PLDMV VIGS vector is a rapid and convenient tool for functional genomic studies in papaya.

中文翻译：

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一种用于木瓜病毒诱导基因沉默的有效木瓜叶扭曲花叶病毒载体

番木瓜 （番木瓜L.) 被认为是热带树木基因组研究的优秀模型，因为它的世代时间短且已测序的基因组小。然而，木瓜的功能基因组研究依赖于费力的遗传转化，因为该物种不存在快速工具。在这里，我们通过修饰木瓜叶片扭曲花叶病毒（PLDMV；属：马铃薯病毒)，PLDMV-E，进入稳定的基于灵活克隆 (NC) 的 PLDMV 载体 pPLDMV-NC，在大肠杆菌. 基因沉默的目标片段可以很容易地克隆到 pPLDMV-NC 中，而无需多次消化和连接步骤。使用这个 PLDMV VIGS 系统，我们沉默并表征了木瓜中的五个内源基因，包括两个常见的 VIGS 标记基因，即，八氢番茄红素去饱和酶,镁螯合酶 H 亚基, 推定赤霉素 (GA)-不敏感 DWARF1A和1B编码 GA 受体；和细胞色素 P450 基因CYP83B1，它编码参与苄基硫代葡萄糖苷生物合成的关键酶。结果表明，我们新开发的 PLDMV VIGS 载体是一种快速便捷的木瓜功能基因组研究工具。