复制酶基因介导的 RNA 沉默机制赋予番茄(Lycopersicon esculentum Mill.)

IF 1.7 3区 农林科学 Q2 AGRONOMY
Karthikeyan Gandhi, Suganyadevi Murugesan, Rajamanickam Suppaiah
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引用次数: 0

摘要

落花生花蕾坏死病毒(GBNV)是正瘤病毒属的一种,是导致番茄花蕾枯萎病的最具破坏性的病原体,在印度造成了巨大的作物损失。目前的管理策略依赖于使用耐病毒的栽培品种、控制昆虫媒介和其他栽培措施。在田间条件下,这些方法无法有效减少病害。可以通过 RNA 沉默来实现控制,这种方法可以调节目标基因的同源特异性降解,从而减少病毒的繁殖。在本研究中,从泰米尔纳德邦的不同地区采集了受病毒感染的番茄植株样本,并使用特异于 GBNV 的多克隆抗体通过 DAC-ELISA 确认了感染情况。病毒接种体在当地的病害宿主豇豆(Vigna unguiculata)上繁殖,导致接种的主叶片上产生萎黄和坏死斑点。我们的研究结果表明,利用 PCR 鉴定出的复制酶基因的完整核苷酸序列与其他 GBNV 分离物的相同度为 94.7% 至 97.7%。为了研究病毒抑制机制,我们利用 GBNV 复制酶基因的保守序列开发了一种有效的 RNAi 构建物。在 pHANNIBAL 载体中插入了一个 3674 bp 的 hpRNA 盒,其中包括 357 bp 的有义和反义片段以及侧翼序列。通过使用 nptII 和 Rep 基因进行 PCR 分析,以及使用 DIG 发光检测试剂盒进行点印迹杂交,评估了所培育的推定转化体中是否存在转基因。复制酶 hpRNA 构建体的表达表明,在人工接种 GBNV 后,症状的发展有所减弱。使用 DAC-ELISA 对转基因番茄植株进行的进一步分析证实了病毒滴度的降低。我们认为,用复制酶基因的保守序列构建的 RNAi 构建物显示了一种基因沉默机制,这从推测的转基因品系中病毒积累的减少可以得到证明。
本文章由计算机程序翻译,如有差异,请以英文原文为准。

Replicase gene mediated RNA silencing mechanism confers resistance against groundnut bud necrosis virus in tomato (Lycopersicon esculentum Mill.)

Replicase gene mediated RNA silencing mechanism confers resistance against groundnut bud necrosis virus in tomato (Lycopersicon esculentum Mill.)

Groundnut bud necrosis virus (GBNV), a member of the genus Orthotospovirus, is the most devastating pathogen causing bud blight of tomato and causes substantial crop losses in India. Current management strategies rely upon the use of virus tolerant cultivars, control of insect vectors, and other cultural practices. Under field conditions, these methods are ineffective in reducing the disease. Control can be achieved with RNA silencing, which regulates the homologous specific degradation of targeted genes, resulting in reduced virus multiplication. In the present study, virus infected tomato plant samples were collected from different parts of Tamil Nadu, and infection was confirmed through DAC-ELISA using a polyclonal antibody specific to GBNV. The virus inoculum was propagated on the local lesion host, cowpea (Vigna unguiculata), resulting in the production of chlorotic and necrotic spots on inoculated primary leaves. Our results demonstrated that the complete nucleotide sequence of the replicase gene identified using PCR shared an identity of 94.7 to 97.7% with other isolates of GBNV. To investigate the virus suppression mechanism, an effective RNAi construct was developed with the conserved sequence of the replicase gene for GBNV. A 3674 bp hpRNA cassette, comprising the sense and antisense fragments of 357 bp along with the flanking sequence, inserted in a pHANNIBAL vector, generated transgenic tomato plants using shoot apical meristem explants through Agrobacterium harboring the gene construct. The presence of the transgene in the developed putative transformants was assessed by PCR analysis using nptII and Rep genes and dot blot hybridization using a DIG luminescent detection kit. The expression of the replicase hpRNA construct revealed reduced symptom development upon artificial inoculation of GBNV. Further analysis of the transgenic tomato plants using DAC-ELISA confirmed the reduced level of virus titer. We propose that the RNAi construct, established with a conserved sequence of the replicase gene, showed a gene silencing mechanism as evidenced by reduced virus accumulation in putative transgenic lines, and this could be used as an effective strategy in the management of GNBV in tomato.

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来源期刊
European Journal of Plant Pathology
European Journal of Plant Pathology 农林科学-农艺学
CiteScore
4.20
自引率
5.60%
发文量
183
审稿时长
3 months
期刊介绍: The European Journal of Plant Pathology is an international journal publishing original articles in English dealing with fundamental and applied aspects of plant pathology; considering disease in agricultural and horticultural crops, forestry, and in natural plant populations. The types of articles published are :Original Research at the molecular, physiological, whole-plant and population levels; Mini-reviews on topics which are timely and of global rather than national or regional significance; Short Communications for important research findings that can be presented in an abbreviated format; and Letters-to-the-Editor, where these raise issues related to articles previously published in the journal. Submissions relating to disease vector biology and integrated crop protection are welcome. However, routine screenings of plant protection products, varietal trials for disease resistance, and biological control agents are not published in the journal unless framed in the context of strategic approaches to disease management.
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