软体动物毛蚶RNA干扰技术的应用研究

Research of RNA Interference Technique in Mollusk Scapharca kagoshimensis

为了在软体动物中建立RNA干扰技术,以用于毛蚶基因功能研究。通过体外转录技术合成双链RNA,并将其注射毛蚶干扰目标基因的表达。肌肉注射双链RNA48h后取样,用RT-PCR技术检测目标基因的表达变化,以确定干扰的有效性。结果显示:各目标基因的表达均能被各自特异的双链RNA有效干扰而降低。在注射双链RNA后不同时间点,即12、48、72、96、120h,取样检测目标基因的时序表达,以确定目标基因表达被干扰的时间效应。发现目标基因的表达在48h即能有效降低,而且有效时间能持续至120h以上。对注射双链RNA的动物个体,于48h取样检测不同组织的目标基因表达,发现毛蚶的RNA干扰效应能系统性地在不同组织发生,表明干扰效应能在不同组织之间进行传递。因此,本研究报道了一种稳定的毛蚶RNA干扰技术,为在毛蚶中进行基因功能鉴定和抗病毒研究提供了一个有效的分子生物学的方法。

To establish a RNA interference technique in Scapharca kagoshimensis for usage in research of gene function, double strand RNA was synthesized using in vitro transcription. The genes examined here using RT-PCR technique for determining the validity of RNA interference could be effectively knocked down at 48 h post injection of double strand RNA. The shortest effective time of RNA interference was found to be 48 h post injection when the gene expression was analyzed at different time points, i.e. 12, 48, 72, 96, 120 h after injection of dsRNA for examining the temporal effect of RNA interference, and the effective time could last up to 120 h. Moreover, the decrease of gene expression could be observed in different tissues, indicating that RNA interference could be induced systemically and the interference effect could be transported among different tissues. Here, we reported a stable RNA interference technology which could provided an effective molecular biological approach for the identification of gene function and antiviral research.