瞬时表达靶向TMV外壳蛋白基因的siRNA能干扰病毒侵染

Transiently expressed siRNAs targeting coat protein gene of Tobacco mosaic virus interferes with the virus infection

RNA干扰(RNA interference,RNAi)是与内源性mRNA编码区某段序列同源的双链RNA导入细胞后,该mRNA发生特异性降解,从而导致该基因表达沉默的现象。小干扰RNA(small interfering RNA,siRNA)作为RNAi途径的重要中介, 已被广泛应用于动、植物抗病毒治疗研究。本文以烟草花叶病毒(Tobacco mosaic virus,TMV)外壳蛋白基因为靶位,设计合成表达小干扰RNA的寡核苷酸,亚克隆到植物双元表达载体pBI121中,直接转化根癌农杆菌。通过根癌农杆菌介导的瞬时表达法,研究了同源于TMV外壳蛋白的siRNA对TMV侵染的干扰作用。结果表明,瞬时表达的siRNA能够特异性干扰TMV侵染。含有重组表达载体pBI121／siRNA的根癌农杆菌渗入普通烟植株,在TMV接种后14 d其上部叶片没有表现典型的花叶症状。对这些叶片进行Northern杂交试验也没有检测到TMV病毒的RNA积累或仅有很少量的积累。在枯斑寄主心叶烟上,siRNA的瞬时表达可使TMV侵染后的枯斑数明显减少,甚至不产生枯斑。此外,同源于TMV外壳蛋白的siRNA瞬时表达对非同源的黄瓜花叶病毒(Cucumber mosaic virus,CMV)没有抑制作用,表明siRNA的干扰作用具有高度的同源依赖性。

Double-strand RNA （dsRNA） induced sequence-specific post-transcriptional gene silencing was known as RNA interference （RNAi）. siRNA, an important intermediate of the RNA-interference pathway, are very effective in antiviral therapy in many organisms including mammals and plants. Oligonucleotides expressed siRNA were designed and synthesized according to the coat protein （CP） gene of Tobacco mosaic virus （TMV） and subcloned to the binary vector pBI121. The interference effects of siRNA targeting CP gene on TMV infection were studied by Agrobacterium-mediated transient expression system. Our results showed that transiently expressed siRNA could specifically interfere with TMV infection. Fourteen days after inoculation with TMV, the upper leaves of Nicotiana tobacum infiltrated with Agrobacterium tumefaciens cultures containing pBI121/siRNA were free of mosaic symptom. Northern blot assays also showed there were no or much less TMV RNA accumulation in these leaves. As for local lesion host, Nicotiana glutinosa, transiently expressed siRNA could reduce the lesion number resulting from TMV infection. In addition, transiently expressed siRNA targeting CP gene of TMV was unable to inhibit Cucumber mosaic virus （ CMV）, which indicated that the interference effect of siRNA was strictly dependent on the specific nucleotide sequence. Therefore, we supposed that the combination of siRNA technique and Agrobacterium-mediated transient expression system could be used as an antiviral treatment or rapid diagnostic tool in plants.