Mitra A, Higgins D W, Langenberg W G, Nie H, Sengupta D N, Silverman R H
Center for Biotechnology and Department of Plant Pathology, University of Nebraska, Lincoln 68583-0722, USA.
Proc Natl Acad Sci U S A. 1996 Jun 25;93(13):6780-5. doi: 10.1073/pnas.93.13.6780.
Resistance to virus infections in higher vertebrates is mediated in part through catalysis of RNA decay by the, interferon-regulated 2-5A system. A functional 2-5A system requires two enzymes, a 2-5A synthetase that produces 5'-phosphorylated, 2',5'-linked oligoadenylates (2-5A) in response to double-stranded RNA, and the 2-5A-dependent RNase L. We have coexpressed these human enzymes in transgenic tobacco plants by using a single plasmid containing the cDNAs for both human RNase L and a low molecular weight form of human 2-5A synthetase under control of different, constitutive promoters. Expression of the human cDNAs in the transgenic plants was demonstrated from Northern blots, by specific enzyme assays, and by immunodetection (for RNase L). Infection of leaves, detached or in planta, of the coexpressing transgenic plants by tobacco mosaic virus, alfalfa [correction of alfafa] mosaic virus, or tobacco etch virus resulted in necrotic lesions. In contrast, leaves expressing 2-5A synthetase or RNase L alone and leaves containing the plasmid vector alone produced typical systemic infections. While alfalfa mosaic virus produced lesions only in the inoculated leaves regardless of the concentration of virus in the inoculum, high, but not low, levels of tobacco etch virus inoculum resulted in escape of virus to uninoculated leaves. Nevertheless, there was a substantial reduction of tobacco etch virus yield as measured by ELISA assay in the coexpressing transgenic plants. These results indicate that expression of a mammalian 2-5A system in plants provides resistance to virus infections.
高等脊椎动物对病毒感染的抗性部分是通过干扰素调节的2-5A系统催化RNA降解来介导的。一个功能性的2-5A系统需要两种酶，一种是2-5A合成酶，它能响应双链RNA产生5'-磷酸化的2',5'-连接的寡聚腺苷酸（2-5A），另一种是依赖2-5A的核糖核酸酶L。我们通过使用一个单一质粒在转基因烟草植物中共表达了这些人类酶，该质粒在不同的组成型启动子控制下含有人类核糖核酸酶L和低分子量形式的人类2-5A合成酶的cDNA。通过Northern印迹、特异性酶分析和免疫检测（针对核糖核酸酶L）证实了转基因植物中人类cDNA的表达。用烟草花叶病毒、苜蓿花叶病毒或烟草蚀纹病毒感染共表达转基因植物的离体或整株叶片，会导致坏死斑。相比之下，单独表达2-5A合成酶或核糖核酸酶L的叶片以及仅含有质粒载体的叶片产生典型的系统感染。虽然苜蓿花叶病毒无论接种物中病毒浓度如何，仅在接种叶片中产生病斑，但高浓度而非低浓度的烟草蚀纹病毒接种物会导致病毒扩散到未接种的叶片。然而，通过ELISA检测发现，在共表达转基因植物中烟草蚀纹病毒的产量大幅降低。这些结果表明，在植物中表达哺乳动物的2-5A系统可提供对病毒感染的抗性。
