RNA interference (RNAi) shows significant effectiveness in conferring resistance to viral infections in various plants. The current study was conducted to develop transgenic tomato ( cv. Rio Grande) lines expressing a 320 bp conserved inverted region of the integral 35S promoter region. The molecular cloning was done for the construction of expression vectors in a hairpin structure by the (TSWV) species , silencing suppressor () gene. It contains both sense and antisense orientation in a binary vector pFGC5941. Short hairpin RNA (shRNA) structures were employed to reduce the possibility of inducing endogenous non-specific antiviral responses, which usually target longer double-stranded RNAs (dsRNAs). To confirm the transgenic plants, molecular analysis was performed using TSWV gene-specific primers (TSWV-). Based on the EHA105 strain and the pFGC5941 vector carrying and genes in the T-DNA region, the results validate the insertion of kanamycin resistance into the regenerated transgenic plants. The virus source harboring TSWV was used to inoculate putative transgenic plants for evaluating the confirmation of successful transformation strategy for inducing virus resistance. Quantitative real-time polymerase chain reaction (qRT-PCR) assays were performed by using qRT- primer pairs to determine the relative gene expression of all constructs (TSWV- both sense and antisense orientation) after inoculations. In comparison to the control groups, which contained wild type control (non-transgenic tomato plant), empty pFGC5941 vector and the pFGC5941+sense construct, the expression of the gene was noticeably reduced in the full clone (pFGC5941+sense + antisense) construct. For the validation of RNAi effectiveness, plants were also inoculated with viral constructs. Specifically, they displayed severe symptoms of TSWV in plants containing the pFGC5941+sense, pFGC5941 empty vector, and in wild-type tomato plants that are correlated with bioassay results. The lack of TSWV symptoms validated the hypothesis after careful observation of the expression of genes in terms of pathogenicity. This research demonstrates the RNA interference effectiveness by targeting gene as a reliable technique for achieving long lasting antiviral protection in tomatoes. The findings have practical relevance and the potential to be a tool for limiting tomato crop losses caused by TSWV worldwide.

中文翻译：

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通过植物介导的 RNAi 方法敲低正孢病毒源性沉默抑制基因可诱导番茄病毒抗性

RNA 干扰 (RNAi) 在赋予多种植物抗病毒感染方面显示出显着的有效性。目前的研究旨在开发表达完整 35S 启动子区域的 320 bp 保守反向区域的转基因番茄 (cv. Rio Grande) 品系。分子克隆是为了通过（TSWV）物种的沉默抑制（）基因构建发夹结构的表达载体。它在二元载体 pFGC5941 中包含有义和反义方向。采用短发夹 RNA (shRNA) 结构来降低诱导内源性非特异性抗病毒反应的可能性，这种反应通常针对较长的双链 RNA (dsRNA)。为了确认转基因植物，使用 TSWV 基因特异性引物 (TSWV-) 进行分子分析。基于EHA105菌株和在T-DNA区域携带基因的pFGC5941载体，结果验证了卡那霉素抗性插入到再生的转基因植物中。使用含有 TSWV 的病毒源接种假定的转基因植物，以评估诱导病毒抗性的成功转化策略的确认。通过使用 qRT-引物对进行定量实时聚合酶链反应（qRT-PCR）测定，以确定接种后所有构建体（TSWV-有义和反义方向）的相对基因表达。与包含野生型对照（非转基因番茄植物）、空pFGC5941载体和pFGC5941+有义构建体的对照组相比，全克隆（pFGC5941+有义+反义）构建体中基因的表达显着降低。为了验证 RNAi 的有效性，植物还接种了病毒构建体。具体来说，他们在含有 pFGC5941+sense、pFGC5941 空载体的植物以及与生物测定结果相关的野生型番茄植物中表现出严重的 TSWV 症状。在仔细观察致病性基因的表达后，TSWV 症状的缺乏验证了这一假设。这项研究证明了通过靶向基因进行 RNA 干扰的有效性，这是一种在番茄中实现持久抗病毒保护的可靠技术。研究结果具有实际意义，并有可能成为限制全球 TSWV 造成的番茄作物损失的工具。