Mirauti Andrea, Tran Phu-Tri, Citovsky Vitaly
Department of Biochemistry and Cell Biology, State University of New York, Stony Brook, NY 11794-5215, USA.
Heliyon. 2023 Sep 5;9(9):e19855. doi: 10.1016/j.heliyon.2023.e19855. eCollection 2023 Sep.
Transgenic expression of proteins in plants is central to research and biotechnology, and, often, it is desirable to obtain this expression without altering the nuclear or plastid genomes. Thus, expression vectors based on plant viruses that infect multiple cells are useful; furthermore, they are also advantageous for studies of the life cycle of the virus itself. Here, we report the development of an expression vector based on a (TVCV), a tobamovirus known to easily infect two model plants, , and . Avoiding restriction digestion, we utilized a restriction-ligation-independent cloning approach to construct an infectious cDNA clone of TVCV from the viral RNA and then to convert this clone to a gene expression vector adapted for Gateway-based recombination cloning for transgene insertion. The functionality of the resulting vector, designated pTVCV-DEST, was validated by the expression of an autofluorescent reporter transgene following agroinoculation of the target plant.
植物中蛋白质的转基因表达是研究和生物技术的核心，而且通常希望在不改变核基因组或质体基因组的情况下实现这种表达。因此，基于能感染多个细胞的植物病毒的表达载体很有用；此外，它们对于病毒自身生命周期的研究也具有优势。在此，我们报道了一种基于番茄病毒（TVCV）构建的表达载体，TVCV是一种烟草花叶病毒属病毒，已知它能轻易感染两种模式植物，即本氏烟草和番茄。我们避免使用限制性酶切，而是采用了一种不依赖限制性酶切连接的克隆方法，从病毒RNA构建了TVCV的感染性cDNA克隆，然后将该克隆转化为适合基于Gateway重组克隆的基因表达载体，用于转基因插入。通过在目标植物上进行农杆菌接种后，一个自发荧光报告转基因的表达，验证了所得载体（命名为pTVCV-DEST）的功能。
