Shivprasad S, Pogue G P, Lewandowski D J, Hidalgo J, Donson J, Grill L K, Dawson W O
Department of Plant Pathology, University of Florida, Lake Alfred, Florida, 33850, USA.
Virology. 1999 Mar 15;255(2):312-23. doi: 10.1006/viro.1998.9579.
A series of tobacco mosaic virus (TMV)-based hybrid vectors for transient gene expression were constructed with similar designs but differing in the source of heterologous tobamovirus sequence: Odontoglossum ringspot virus, tobacco mild green mosaic virus variants U2 and U5, tomato mosaic virus, and sunn-hemp mosaic virus. These vectors contained a heterologous coat protein subgenomic mRNA promoter and coat protein open reading frame (ORF) and either TMV or heterologous 3' nontranslated region. The foreign ORF, from the jellyfish green fluorescent protein (GFP) gene, was transcribed from the native TMV coat protein subgenomic mRNA promoter, which extended into the coat protein ORF. The presence of an in-frame stop codon within the GFP mRNA leader and the choice of sequence of GFP ORFs substantially affected translational efficiency. However, the major regulatory component of gene expression in these vectors appeared to be transcriptional rather than translational. There was an inverse relationship between expression of GFP and the heterologous coat protein genes that was reflected in accumulation of the respective mRNAs and proteins. The most effective vector in this series (30B) contained sequences encoding the coat protein subgenomic mRNA promoter, coat protein ORF, and 3' nontranslated region from tobacco mild green mosaic virus U5. Expressed from 30B, GFP accumulated up to 10% of total soluble protein in leaves.
构建了一系列基于烟草花叶病毒（TMV）的用于瞬时基因表达的杂交载体，它们设计相似，但异源烟草花叶病毒序列来源不同：齿舌兰环斑病毒、烟草轻绿花叶病毒变种U2和U5、番茄花叶病毒以及印度麻花叶病毒。这些载体包含一个异源外壳蛋白亚基因组mRNA启动子和外壳蛋白开放阅读框（ORF），以及TMV或异源3'非翻译区。来自水母绿色荧光蛋白（GFP）基因的外源ORF由天然TMV外壳蛋白亚基因组mRNA启动子转录，该启动子延伸至外壳蛋白ORF内。GFP mRNA前导序列中框内终止密码子的存在以及GFP ORF序列的选择对翻译效率有显著影响。然而，这些载体中基因表达的主要调控成分似乎是转录而非翻译。GFP和异源外壳蛋白基因的表达之间存在反比关系，这在各自mRNA和蛋白质的积累中得到体现。该系列中最有效的载体（30B）包含来自烟草轻绿花叶病毒U5的编码外壳蛋白亚基因组mRNA启动子、外壳蛋白ORF和3'非翻译区的序列。由30B表达的GFP在叶片中积累量高达总可溶性蛋白的10%。
