Kim Jihyun, Park Minkyu, Jeong Eun Soo, Lee Je Min, Choi Doil
Department of Plant Science, Plant Genomics and Breeding Institute, College of Agriculture and Life Sciences, Seoul National University, 1 Gwanak-ro, Gwanak-gu, Seoul, 08826 Korea.
Department of Plant Science, Plant Genomics and Breeding Institute, College of Agriculture and Life Sciences, Seoul National University, 1 Gwanak-ro, Gwanak-gu, Seoul, 08826 Korea ; Department of Genetics, University of Georgia, Athens, GA 30602-7223 USA.
Plant Methods. 2017 Jan 3;13:3. doi: 10.1186/s13007-016-0151-5. eCollection 2017.
Virus-induced gene silencing (VIGS) has become a powerful tool for post-genomic technology in plant species. This is important, especially in select plants, such as the pepper plant, that are recalcitrant to -mediated transformation. Although VIGS in plants has been widely employed as a powerful tool for functional genomics, scattering phenotypic effects by uneven gene silencing has been implemented in order to overcome challenges in experiments with fruit tissues.
We improved the VIGS system based on the tobacco rattle virus (TRV) containing the MYB transcription factor, which is the genetic determinant of purple colored- or anthocyanin-rich pepper. Silencing of endogenous in the anthocyanin-rich pepper with the modified TRV vector for ligation-independent cloning (LIC) lacked purple pigment in its leaves, flowers, and fruits. Infection with TRV-LIC containing a tandem construct of and () resulted in a typical photobleaching event in leaves without the purple pigment, whereas silencing of led to the presence of photobleached and purple-colored leaves. Cosilencing of endogenous and in fruits resulted in decreased levels of capsaicin and dihydrocapsaicin as assessed by high performance liquid chromatography analysis coupled with the absence of the purple pigment in fruits.
VIGS with tandem constructs harboring as a visible reporter in anthocyanin-rich pepper plants can facilitate the application of functional genomics in the study of metabolic pathways and fruit biology.
病毒诱导的基因沉默（VIGS）已成为植物后基因组技术的一种强大工具。这一点很重要，尤其是在一些对介导转化具有抗性的特定植物中，比如辣椒植株。尽管植物中的VIGS已被广泛用作功能基因组学的强大工具，但为了克服果实组织实验中的挑战，已采用通过不均匀基因沉默来分散表型效应的方法。
我们改进了基于烟草脆裂病毒（TRV）的VIGS系统，该病毒含有MYB转录因子，它是紫色或富含花青素辣椒的遗传决定因素。用用于不依赖连接克隆（LIC）的改良TRV载体沉默富含花青素辣椒中的内源性基因，其叶片、花朵和果实中缺乏紫色色素。用含有和串联构建体（）的TRV-LIC感染导致叶片出现典型的光漂白现象且无紫色色素，而沉默则导致出现光漂白和紫色叶片。通过高效液相色谱分析评估，果实中内源性基因和基因的共沉默导致辣椒素和二氢辣椒素水平降低，同时果实中没有紫色色素。
在富含花青素的辣椒植株中，将作为可见报告基因的串联构建体用于VIGS，有助于功能基因组学在代谢途径和果实生物学研究中的应用。
