Sandra Nagamani, Jailani A Abdul Kader, Jain Rakesh Kumar, Mandal Bikash
Division of Seed Science and Technology, Indian Agricultural Research Institute, New Delhi, 110012, India.
Advanced Centre for Plant Virology, Division of Plant Pathology, Indian Agricultural Research Institute, New Delhi, 110012, India.
Mol Biotechnol. 2019 Mar;61(3):181-190. doi: 10.1007/s12033-018-0147-7.
Plant virus-based vectors provide attractive and valuable tools for rapid production of recombinant protein in large quantities as they produce systemic infections in differentiated plant tissues. In the present study, we engineered the Soybean yellow mottle mosaic virus (SYMMV) as a gene expression vector which is a promising candidate for systemic expression of foreign proteins in French bean plants. Full virus vector strategy was exploited for insertion of foreign gene by inserting MCS through PCR in the circular pJET-SYMMV clone. To examine the ability of the SYMMV vector system, GFP gene was cloned after the start codon of coat protein (CP) so that its expression was driven by the SYMMV-CP subgenomic promoter. When in vitro run off SYMMV-GFP transcript was mechanically inoculated to French bean leaves, good level of GFP expression was observed through confocal microscopy up to 40 dpi. Expression of heterologous protein was also confirmed through ISEM, DAC-ELISA and RT-PCR with specific primers at 20 dpi. The recombinant SYMMV construct was stable in in vitro runoff transcript inoculated plants but the inserted GFP was lost in progeny virion inoculated plants. The system developed here will be useful for further studies of SYMMV gene functions and exploitation of SYMMV as a gene expression vector.
基于植物病毒的载体为在分化的植物组织中产生系统感染从而大量快速生产重组蛋白提供了有吸引力且有价值的工具。在本研究中，我们构建了大豆黄斑花叶病毒（SYMMV）作为基因表达载体，它是在菜豆植株中系统表达外源蛋白的一个有前景的候选载体。通过在环状pJET - SYMMV克隆中利用PCR插入多克隆位点（MCS），采用全病毒载体策略来插入外源基因。为了检测SYMMV载体系统的能力，在外壳蛋白（CP）的起始密码子之后克隆了绿色荧光蛋白（GFP）基因，使其表达由SYMMV - CP亚基因组启动子驱动。当将体外转录的SYMMV - GFP转录本机械接种到菜豆叶片上时，通过共聚焦显微镜观察到在接种后40天内GFP表达水平良好。在接种后20天，还通过免疫扫描电镜（ISEM）、双抗体夹心酶联免疫吸附测定（DAC - ELISA）和使用特异性引物的逆转录 - 聚合酶链反应（RT - PCR）证实了异源蛋白的表达。重组SYMMV构建体在体外转录本接种的植株中是稳定的，但在子代病毒粒子接种的植株中插入的GFP丢失了。这里开发的系统将有助于进一步研究SYMMV基因功能以及将SYMMV用作基因表达载体。
