Background The raised demand of cannabis as a medicinal plant in recent years led to an increased interest in understanding the biosynthetic routes of cannabis metabolites. Since there is no established protocol to generate stable gene knockouts in cannabis, the use of a virus-induced gene silencing (VIGS) method, resulting in a gene knockdown, to study gene functions is desirable. Results For this, a computational approach was employed to analyze the Cannabis sativa L. transcriptomic and genomic resources. Reporter genes expected to give rise to easily scorable phenotypes upon silencing, i.e. the phytoene desaturase (PDS) and magnesium chelatase subunit I (ChlI), were identified in C. sativa. Subsequently, the targets of specific small interfering RNAs (siRNAs) and silencing fragments were predicted and tested in a post-transcriptional gene silencing (PTGS) approach. Here we show for the first time a gene knockdown in C. sativa using the Cotton leaf crumple virus (CLCrV) in a silencing vector system. Plants transiently transformed with the Agrobacterium tumefaciens strain AGL1, carrying the VIGS-vectors, showed the desired phenotypes, spotted bleaching of the leaves. The successful knockdown of the genes was additionally validated by quantitative PCR resulting in reduced expression of transcripts from 70 to 73% for ChlI and PDS, respectively. This is accompanied with the reduction of the chlorophyll a and carotenoid content, respectively. In summary, the data clearly demonstrate the potential for functional gene studies in cannabis using the CLCrV-based vector system. Conclusions The applied VIGS-method can be used for reverse genetic studies in C. sativa to identify unknown gene functions. This will gain deeper inside into unknown biosynthetic routes and will help to close the gap between available genomic data and biochemical information of this important medicinal plant.

中文翻译：

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大麻中病毒诱导的基因沉默（VIGS）。

背景 近年来，大麻作为药用植物的需求不断增加，导致人们对了解大麻代谢物的生物合成途径越来越感兴趣。由于没有既定的方案在大麻中产生稳定的基因敲除，因此需要使用病毒诱导的基因沉默（VIGS）方法，导致基因敲低，以研究基因功能。结果 为此，采用计算方法来分析大麻转录组和基因组资源。在苜蓿中鉴定了预期在沉默后产生容易评分的表型的报告基因，即八氢番茄红素去饱和酶(PDS)和镁螯合酶亚基I(ChlI)。随后，通过转录后基因沉默 (PTGS) 方法预测和测试了特定小干扰 RNA (siRNA) 和沉默片段的靶标。在这里，我们首次在沉默载体系统中使用棉叶皱缩病毒（CLCrV）显示了苜蓿中的基因敲低。用携带 VIGS 载体的根癌农杆菌菌株 AGL1 瞬时转化的植物显示出所需的表型，叶子出现斑点漂白。基因的成功敲低还通过定量 PCR 进行了验证，导致 ChlI 和 PDS 的转录物表达分别从 70% 降低至 73%。这分别伴随着叶绿素 a 和类胡萝卜素含量的减少。总之，数据清楚地证明了使用基于 CLCrV 的载体系统进行大麻功能基因研究的潜力。结论 VIGS方法可用于苜蓿的反向遗传学研究，以鉴定未知基因的功能。这将更深入地了解未知的生物合成途径，并将有助于缩小这种重要药用植物的可用基因组数据和生化信息之间的差距。