中文摘要：番茄斑萎病毒（TSWV）是广泛的大田作物和园艺作物上一种重要的经济性病毒病原体。作者开发了一个抵抗TSWV的以核蛋白（N）和沉默抑制子（NSs）基因为靶标的人工microRNA (amiRNA)策略。研究结果表明：缩短的173-nt athmiR159a backbone足以表达amiRNAs，并且12-13位点的错配并不影响amiRNAs的表达或抗性的授予。研究还表明：目标基因的选择和定位效果对基于amiRNA方法的抗性引进至关重要。这些发现开启了利用amiRNA方法发展对抗番茄斑萎病毒属病毒以及其他病毒广谱抗性的可能性。
外文摘要：Tomato spotted wilt virus (TSWV) is an economically important viral pathogen of a wide range of field and horticultural crops. We developed an artificial microRNA (amiRNA) strategy against TSWV, targeting the nucleoprotein (N) and silencing suppressor (NSs) genes. The amiRNA constructs replaced the natural miRNA in a shortened Arabidopsis 173-nucleotide (nt) miR159a precursor backbone (athmiR159a) without the stem base extending beyond the miR/miR* duplex. Further, each amiRNA was modified to contain a mismatch (wobble) sequence at nucleotide position 12 and 13 on the complementary strand amiRNA*, mimicking the endogenous miR159a sequence structure. Transient expression in Nicotiana benthamiana demonstrated that the introduction of a wobble sequence did not alter amiRNA expression levels. Following challenge inoculation with TSWV, plants expressing N-specific amiRNAs with or without the wobble remained asymptomatic and were negative for TSWV by ELISA. In contrast, plants expressing the NSs-specific amiRNAs were symptomatic and accumulated high levels of TSWV. Similar findings were obtained in stably transformed Nicotiana tabacum plants. Our results show that a shortened 173-nt athmiR159a backbone is sufficient to express amiRNAs and that the presence of mismatch at position 12-13 does not influence amiRNA expression or conferring of resistance. We also show that selection of target gene and positional effect are critical in amiRNA-based approach for introducing resistance. These findings open the possibility of employing the amiRNA approach for broad-spectrum resistance to tospoviruses as well as other viruses.
外文关键词：RNA silencing; Tomato spotted wilt virus; Artificial microRNA; Virus resistance
作者：Mitter, Neena; Zhai, Ying; Bai, Anh Xu; 等
作者单位：华盛顿州立大学
期刊名称：VIRUS RESEARCH
期刊影响因子：2.526
出版年份：2016
出版刊次：1
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