入侵植物薇甘菊Kunth(M.来自南美的micrantha)对生态系统的稳定性和生物多样性构成了重大威胁。然而,目前还缺乏一种有效和经济的控制薇甘菊的方法。RNA干扰(RNAi)已被广泛研究并应用于农业性状改良。喷雾诱导的基因沉默(SIGS)可以产生RNAi沉默效应,而不会对植物基因组引入可遗传的修饰,并且正在成为一种新型的植物保护非转化策略。在这项研究中,选择编码叶绿素a/b结合蛋白的基因作为RNAi的靶标,基于薇甘菊转录组的高通量测序和序列特异性的生物信息学分析。三种类型的RNAi分子,双链RNA,RNAi纳米微球,和短发夹RNA(shRNA),设计并合成了相应的短干扰RNA序列,用于构建SIGS载体,其中每个RNAi分子被转录并提取以喷洒到薇甘菊叶上。而水处理的对照叶仍然是绿色的,用RNAi分子处理的叶子变黄并最终枯萎。实时定量PCR显示,与对照组相比,RNAi处理组的靶基因表达水平明显降低,表明所有三种类型的RNAi除草剂都有效地沉默了内源靶基因,这对薇甘菊的生长至关重要。我们还发现shRNA显示出比其他两种分子更好的沉默效率。一起来看,我们的研究成功设计了三种类型的基于RNAi的除草剂,这些除草剂特异性沉默了内源性靶基因并控制了薇甘菊的生长。此外,我们确定了一个编码叶绿素a/b结合蛋白的基因家族,该蛋白对薇甘菊的生长和发育很重要,可以作为控制薇甘菊传播的潜在靶标。
The invasive plant Mikania micrantha Kunth (M. micrantha) from South America poses a significant threat to the stability and biodiversity of ecosystems. However, an effective and economical method to control M. micrantha is still lacking. RNA interference (RNAi) has been widely studied and applied in agriculture for trait improvement. Spray-induced gene silencing (SIGS) can produce RNAi silencing effects without introducing heritable modifications to the plant genome and is becoming a novel nontransformation strategy for plant protection. In this
study, the genes encoding chlorophyll a/b-binding proteins were selected as targets of RNAi, based on high-throughput sequencing of M. micrantha transcriptome and bioinformatic analyses of sequence specificity. Three types of RNAi molecules, double-stranded RNA, RNAi nanomicrosphere, and short hairpin RNA (shRNA), with their corresponding short interfering RNA sequences were designed and synthesized for SIGS vector construction, from which each RNAi molecule was transcribed and extracted to be sprayed on M. micrantha leaves. Whereas water-treated control leaves remained green, leaves treated with RNAi molecules turned yellow and eventually wilted. Quantitative real-time PCR showed that the expression levels of target genes were significantly reduced in the RNAi-treated groups compared with those of the control, suggesting that all three types of RNAi herbicides effectively silenced the endogenous target genes, which are essential for the growth of M. micrantha. We also found that shRNA showed better silencing efficiency than the other two molecules. Taken together, our
study successfully designed three types of RNAi-based herbicides that specifically silenced endogenous target genes and controlled the growth of M. micrantha. Moreover, we identified a gene family encoding chlorophyll a/b-binding proteins that is important for the growth and development of M. micrantha and could serve as potential targets for controlling the spread of M. micrantha.