【摘要】 RNA干扰作为植物抗病毒免疫的核心机制，其调控网络中SGS3(suppressor of gene silencing)基因在RDR6介导的双链RNA合成过程中扮演关键角色。为深入探究SGS3基因功能，对本氏烟草SGS3基因引入新型多靶点基因沉默系统——PTA(poly-tRNA-amiRNA)技术，并与传统单个amiRNA进行RNAi效率的系统性比较。首先针对SGS3基因的锌指（ZF）、XS和卷曲螺旋（coiled coil,CC）等三个结构域各设计合成一个特异性amiRNA，通过分子克隆技术构建pBI121-SGS3-amiRNA1/2/3重组表达载体。利用农杆菌介导的瞬时表达系统，对三个重组载体进行功能验证，结果显示amiRNA2对SGS3基因的沉默效率显著高于其他两个靶点。基于此，采用Golden Gate克隆技术，将上述三个amiRNA与三个tRNA串联组装，成功构建PTA-SGS3表达盒，并整合至pBI121植物表达载体。通过与携带绿色荧光蛋白标记的大豆花叶病毒侵染性克隆SMV-GFP共侵染本氏烟草，系统检测PTA系统的RNAi效率及抗病毒活性。RTqPCR分析表明，相较于单一amiRNA,PTA系统可使SGS3基因沉默效率提升44.8%，显著增强靶基因沉默效果。通过GFP荧光强度定量分析及DAB组织化学染色实验，发现经PTA表达盒侵染的本氏烟草中，SMV病毒积累量较单amiRNA处理组增加50.6%。更多还原

【Abstract】 RNA interference is a core mechanism of antiviral immunity in plants. The SGS3(suppressor of gene silencing) gene plays a crucial role in the regulatory network of RNA interference during the RDR6-mediated synthesis of double-stranded RNA. To further investigate the function of the SGS3 gene, this paper introduced a novel multi-target gene silencing system—PTA(poly-tRNA-amiRNA) technology—into the SGS3 gene of Nicotiana benthamiana and conducted a systematic comparison of RNAi efficiency with traditional single amiRNA. First, the paper designed and synthesized a specific amiRNA targeting three structural domains of the SGS3 gene: zinc finger(ZF), XS, and coiled coil(CC), respectively. It constructed recombinant expression vectors pBI121-SGS3-amiRNA1/2/3 using molecular cloning techniques. Employing an Agrobacterium-mediated transient expression system, the paper performed functional validation of the three recombinant cassettes. The results show that amiRNA2 exhibits significantly higher silencing efficiency of the SGS3 gene compared to the other two targets. Based on this, Golden Gate cloning technology was utilized to assemble the three aforementioned amiRNAs with three tRNAs in tandem, successfully constructing the PTA-SGS3 expression cassette, which was then integrated into the pBI121 plant expression vector. By co-infecting N. benthamiana with Agrobacterium strains harboring the PTA construct and the SMV-GFP infectious clone, which carried a green fluorescent protein marker, the paper systematically assessed the RNAi efficiency and antiviral activity of the PTA system. RT-qPCR analysis indicates that, compared to a single amiRNA, the PTA system improves the silencing efficiency of the SGS3 gene by 44. 8%, significantly enhancing the silencing effect on the target gene. Quantitative analysis of GFP fluorescence intensity and DAB histochemical staining experiments reveals that N. benthamiana infected with the PTA expression cassettes shows a 50. 6% increase in SMV virus accumulation compared to the single amiRNA treatment group.更多还原