我们专注于双生病毒载体系统,以开发用于植物生物技术的有效载体系统。BegomovirusandCurtovirus,属于双子科,包含一个基因间区(IR)和四个参与复制的基因,包括复制相关蛋白(Rep,C1),转录激活因子(TrAP,C2),和复制增强子(REn,C3)。双生病毒可以使用植物DNA聚合酶和病毒复制相关酶扩增病毒DNA的数千个拷贝,并在高浓度下积累病毒蛋白。在这项研究中,我们优化了基于番茄黄化曲叶病毒(TYLCV)的双生病毒DNA复制子载体,金银花黄脉病毒(HYVV),和温和的卷曲顶部病毒(BMCTV)的快速,重组蛋白的高产植物生产。通过共递送每个复制相关基因和每个IR来确认最佳组合,该IR通过农杆菌浸润在烟草叶片中进行Pontellinaplumata衍生的turbo绿色荧光蛋白(tGFP)基因,从而在3天内实现了有效的复制子扩增和强大的蛋白质生产天。与番茄灌木特技病毒p19蛋白共表达,基因沉默抑制因子,通过稳定mRNA进一步增强tGFP积累。有了这个系统,tGFP蛋白以0.7-1.2mg/g叶片鲜重产生,相当于总可溶性蛋白的6.9-12.1%。这些结果证明了使用双生病毒DNA复制子系统在植物中瞬时表达快速和高水平生产重组蛋白的优势。
We focused on the geminiviral vector systems to develop an efficient vector system for plant biotechnology. Begomoviruses and curtoviruses, which belong to the Geminiviridae family, contain an intergenic region (IR) and four genes involved in replication, including replication-associated protein (Rep, C1), transcriptional activator (TrAP, C2), and replication enhancer (REn, C3). Geminiviruses can amplify thousands of copies of viral DNA using plant DNA polymerase and viral replication-related enzymes and accumulate viral proteins at high concentrations. In this study, we optimized geminiviral DNA replicon vectors based on tomato yellow leaf curl virus (TYLCV), honeysuckle yellow vein virus (HYVV), and mild curly top virus (BMCTV) for the rapid, high-yield plant-based production of recombinant proteins. Confirmation of the optimal combination by co-delivery of each replication-related gene and each IR harboring the Pontellina plumata-derived turbo green fluorescence protein (tGFP) gene via agroinfiltration in Nicotiana benthamiana leaves resulted in efficient replicon amplification and robust protein production within 3 days. Co-expression with the p19 protein of the tomato bush stunt virus, a gene-silencing suppressor, further enhanced tGFP accumulation by stabilizing mRNA. With this system, tGFP protein was produced at 0.7-1.2 mg/g leaf fresh weight, corresponding to 6.9-12.1% in total soluble protein. These results demonstrate the advantages of rapid and high-level production of recombinant proteins using the geminiviral DNA replicon system for transient expression in plants.