Jilin Provincial Key Laboratory of Agricultural Biotechnology, Jilin Academy of Agricultural Sciences, Changchun, 130033, China.
Transgenic Res. 2017 Oct;26(5):665-676. doi: 10.1007/s11248-017-0041-2. Epub 2017 Aug 24.
Viral pathogens, such as soybean mosaic virus (SMV), are a major constraint in soybean production and often cause significant yield loss and quality deterioration. Engineering resistance by RNAi-mediated gene silencing is a powerful strategy for controlling viral diseases. In this study, a 248-bp inverted repeat of the replicase (nuclear inclusion b, NIb) gene was isolated from the SMV SC3 strain, driven by the leaf-specific rbcS2 promoter from Phaseolus vulgaris, and introduced into soybean. The transgenic lines had significantly lower average disease indices (ranging from 2.14 to 12.35) than did the non-transformed (NT) control plants in three consecutive generations, exhibiting a stable and significantly enhanced resistance to the SMV SC3 strain under field conditions. Furthermore, seed mottling did not occur in transgenic seeds, whereas the NT plants produced ~90% mottled seeds. Virus resistance spectrum screening showed that the greenhouse-grown transgenic lines exhibited robust resistance to five SMV strains (SC3, SC7, SC15, SC18, and a recombinant SMV), bean common mosaic virus, and watermelon mosaic virus. Nevertheless, no significantly enhanced resistance to bean pod mottle virus (BPMV, Comovirus) was observed in the transgenic lines relative to their NT counterparts. Consistent with the results of resistance evaluation, the accumulation of each potyvirid (but not of BPMV) was significantly inhibited in the transgenic plants relative to the NT controls as confirmed by quantitative real-time (qRT-PCR) and double antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA). These results demonstrate that robust RNAi-mediated resistance to multiple potyvirids in soybean was conferred by expressing an intron hairpin SMV NIb RNA.
病毒病原体，如大豆花叶病毒（SMV），是大豆生产的主要限制因素，经常导致显著的产量损失和质量恶化。通过 RNAi 介导的基因沉默工程抗性是控制病毒病的一种强大策略。在这项研究中，从 SMV SC3 株中分离出一个 248bp 的复制酶（核包含体 b，NIb）基因的反向重复，由菜豆的叶特异性 rbcS2 启动子驱动，并引入大豆中。在三个连续世代中，与非转化（NT）对照植物相比，转基因系的平均疾病指数（范围从 2.14 到 12.35）显著降低，在田间条件下表现出对 SMV SC3 株的稳定和显著增强的抗性。此外，转基因种子中没有出现种子斑驳，而 NT 植物产生了约 90%的斑驳种子。病毒抗性谱筛选表明，温室种植的转基因系对五种 SMV 株（SC3、SC7、SC15、SC18 和重组 SMV）、菜豆普通花叶病毒和西瓜花叶病毒表现出强大的抗性。然而，与 NT 对照相比，在转基因系中没有观察到对豆荚斑驳病毒（BPMV，Comovirus）的显著增强抗性。与抗性评估结果一致，通过定量实时（qRT-PCR）和双抗体夹心酶联免疫吸附测定（DAS-ELISA）证实，与 NT 对照相比，每个 potyvirid（但不是 BPMV）在转基因植物中的积累显著抑制。这些结果表明，通过表达内含子发夹 SMV NIb RNA，赋予了大豆对多种 potyvirids 的强大 RNAi 介导抗性。
