Beauchemin Chantal, Bougie Véronique, Laliberté Jean-François
Institut National de la Recherche Scientifique, INRS-Institut Armand-Frappier, 531 Boulevard des Prairies Laval, Qué., Canada H7V 1B7.
Virus Res. 2005 Sep;112(1-2):1-8. doi: 10.1016/j.virusres.2005.03.001. Epub 2005 Apr 13.
With the aim of developing a biotechnological tool for the production of foreign proteins in plants, we first engineered an infectious turnip mosaic virus (TuMV) cDNA that contained the jellyfish green fluorescent protein (GFP) gene or the bacterial beta-glucuronidase (GUS) gene (uidA). Two insertion sites were assessed, either between P1 and HCPro cistrons or Pol and CP cistrons. In each construct, the junctions flanking the inserted gene coded for P1 and/or VPg-Pro cleavage recognition site sequences, to produce free GUS or GFP. After transfection by particle bombardment on Brassica perviridis, characteristic symptoms for TuMV infection appeared and Western blot analyses showed that GFP and GUS had been excised from the viral polyprotein. No significant differences in expression level were noticed between the two insertion sites. By RT-PCR, gfp was found to be stable over 30 days post-transfection (dpt) while uidA was gradually lost at 15 dpt. We also created two constructs containing either gene at each insertion sites on the same molecule. Attenuated systemic symptoms were observed after particle bombardment on B. perviridis and Western blot analyses showed that both foreign proteins were produced. Also, the same stability/instability as for the single-gene constructs were observed. These results indicate that it is possible to produce at least two foreign proteins simultaneously in a TuMV-based vector.
为了开发一种用于在植物中生产外源蛋白的生物技术工具，我们首先构建了一种感染性芜菁花叶病毒（TuMV）的cDNA，其包含水母绿色荧光蛋白（GFP）基因或细菌β-葡萄糖醛酸酶（GUS）基因（uidA）。评估了两个插入位点，分别在P1和HCPro顺反子之间或Pol和CP顺反子之间。在每个构建体中，插入基因两侧的接头编码P1和/或VPg-Pro切割识别位点序列，以产生游离的GUS或GFP。通过微粒轰击转染青菜后，出现了TuMV感染的特征性症状，蛋白质免疫印迹分析表明GFP和GUS已从病毒多聚蛋白中切除。两个插入位点之间在表达水平上未观察到显著差异。通过逆转录聚合酶链反应（RT-PCR）发现，转染后30天（dpt）内gfp稳定，而uidA在15 dpt时逐渐丢失。我们还构建了两个构建体，在同一分子的每个插入位点上分别含有这两个基因中的一个。对青菜进行微粒轰击后观察到减弱的系统症状，蛋白质免疫印迹分析表明两种外源蛋白均产生。此外，观察到与单基因构建体相同的稳定性/不稳定性。这些结果表明，在基于TuMV的载体中同时生产至少两种外源蛋白是可能的。
