葡萄叶卷相关病毒1微型复制子的研制及复制所需5'非翻译区序列的遗传分析。

IF 3.1 2区 农林科学 Q2 PLANT SCIENCES
Arunabha Mitra, Sridhar Jarugula, Rayapeti A Naidu
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引用次数: 0

摘要

葡萄叶相关病毒1 (glav -1, Ampelovirus属,Closteroviridae科)具有一个单分子RNA基因组,其大小在18,731和18,946核苷酸(nt)之间变化,以及一个长度在857和922 nt之间变化的5‘非翻译区(5’-NTR)。本研究旨在研究5'-NTR在glav -1复制中的作用。为此,构建了由5′-NTR、复制酶基因模块、绿色荧光蛋白(GFP)报告基因和3′-NTR组成的微型复制子cDNA克隆。通过GFP荧光和Northern blot杂交和RT-qPCR检测,在沉默抑制因子共浸润的烟叶中验证了该微型复制子的功能,并验证了GFP特异性mRNA转录物的存在。当其5'-NTR与glrav1不同遗传变异的相应序列交换时,该迷你复制子保留了功能。相比之下,当GLRaV-1的5'-NTR序列与其他GLRaV物种的相应序列交换时,其迷你复制子没有功能。5‘-NTR的缺失突变表明,基因组5’末端的前32nt对于迷你复制子的复制至关重要。在计算机上预测的5'端32-nt序列的二级结构显示了两个茎环结构和改变这些二级结构的突变或保留二级结构的补偿性突变未能保留迷你复制子的功能,这表明核苷酸序列而不是该基因组区域的任何高阶二级结构,对复制很重要。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
Development of a Minireplicon for Grapevine leafroll-associated virus 1 and Genetic Analyses of Sequences in the 5' Non-Translated Region Required for Replication.

Grapevine leafroll-associated virus 1 (GLRaV-1, genus Ampelovirus, family Closteroviridae) has a monopartite RNA genome with size varying among genetic variants between 18,731 and 18,946 nucleotides (nt) and a 5' non-translated region (5'-NTR), varying in length between 857 and 922 nt. This study was undertaken to examine the role of the 5'-NTR in GLRaV-1 replication. For this purpose, a minireplicon cDNA clone, consisting of the 5'-NTR, the replicase gene module, the green fluorescent protein (GFP) reporter gene, and the 3'-NTR, was constructed. Functionality of the minireplicon was validated by GFP fluorescence and the presence of GFP-specific mRNA transcripts by Northern blot hybridization and RT-qPCR assays in Nicotiana benthamiana leaves agro-coinfiltrated with silencing suppressors. The minireplicon retained functionality when its 5'-NTR was exchanged with corresponding sequences from distinct genetic variants of GLRaV-1. In contrast, the minireplicon of GLRaV-1 was non-functional when its 5'-NTR sequence was swapped with corresponding sequences from other GLRaV species. Deletion mutations in the 5'-NTR indicated that the first 32 nt at the 5'-terminus of the genome are essential for replication of the minireplicon. In silico-predicted secondary structure of the 5'-terminal 32-nt sequence showed two stem-loop structures and mutations that altered these secondary structures or compensatory mutations preserving the secondary structures failed to retain functionality of the minireplicon, suggesting that the nucleotide sequence, rather than any higher order secondary structures in this genomic region, is important for replication.

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来源期刊
Phytopathology
Phytopathology 生物-植物科学
CiteScore
5.90
自引率
9.40%
发文量
505
审稿时长
4-8 weeks
期刊介绍: Phytopathology publishes articles on fundamental research that advances understanding of the nature of plant diseases, the agents that cause them, their spread, the losses they cause, and measures that can be used to control them. Phytopathology considers manuscripts covering all aspects of plant diseases including bacteriology, host-parasite biochemistry and cell biology, biological control, disease control and pest management, description of new pathogen species description of new pathogen species, ecology and population biology, epidemiology, disease etiology, host genetics and resistance, mycology, nematology, plant stress and abiotic disorders, postharvest pathology and mycotoxins, and virology. Papers dealing mainly with taxonomy, such as descriptions of new plant pathogen taxa are acceptable if they include plant disease research results such as pathogenicity, host range, etc. Taxonomic papers that focus on classification, identification, and nomenclature below the subspecies level may also be submitted to Phytopathology.
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