Chen Weiwei, Huang Chaolin, Luo Chenmeng, Zhang Yongshan, Zhang Bin, Xie Zhengqing, Hao Mengyuan, Ling Hua, Cao Gangqiang, Tian Baoming, Wei Fang, Shi Gongyao
Zhengzhou Research Base, State Key Laboratory of Cotton Biology, School of Agricultural Sciences, Zhengzhou University, Zhengzhou 450001, China.
Henan International Joint Laboratory of Crop Gene Resources and Improvements, Zhengzhou University, Zhengzhou 450001, China.
Plants (Basel). 2022 Jul 1;11(13):1765. doi: 10.3390/plants11131765.
The difficulty of genetic transformation has restricted research on functional genomics in cotton. Thus, a rapid and efficient method for gene overexpression that does not rely on genetic transformation is needed. Virus-based vectors offer a reasonable alternative for protein expression, as viruses can infect the host systemically to achieve expression and replication without transgene integration. Previously, a novel four-component barley stripe mosaic virus (BSMV) was reported to overexpress large fragments of target genes in plants over a long period of time, which greatly simplified the study of gene overexpression. However, whether this system can infect cotton and stably overexpress target genes has not yet been studied. In this study, we verified that this new BSMV system can infect cotton through seed imbibition and systemically overexpress large fragments of genes (up to 2340 bp) in cotton. The target gene that was fused with GFP was expressed at a high level in the roots, stems, and cotyledons of cotton seedlings, and stable fluorescence signals were detected in the cotton roots and leaves even after 4 weeks. Based on the BSMV overexpression system, the subcellular localization marker line of endogenous proteins localized in the nucleus, endoplasmic reticulum, plasma membrane, Golgi body, mitochondria, peroxisomes, tonoplast, and plastids were quickly established. The overexpression of a cotton Bile Acid Sodium Symporter GhBASS5 using the BSMV system indicated that GhBASS5 negatively regulated salt tolerance in cotton by transporting Na from underground to the shoots. Furthermore, multiple proteins were co-delivered, enabling co-localization and the study of protein-protein interactions through co-transformation. We also confirmed that the BSMV system can be used to conduct DNA-free gene editing in cotton by delivering split-SpCas9/sgRNA. Ultimately, the present work demonstrated that this BSMV system could be used as an efficient overexpression system for future cotton gene function research.
遗传转化的困难限制了棉花功能基因组学的研究。因此，需要一种不依赖遗传转化的快速高效的基因过表达方法。基于病毒的载体为蛋白质表达提供了一种合理的替代方案，因为病毒可以系统地感染宿主以实现表达和复制，而无需转基因整合。此前，据报道一种新型的四组分大麦条纹花叶病毒（BSMV）能够在植物中长时间过表达目标基因的大片段，这大大简化了基因过表达的研究。然而，该系统是否能感染棉花并稳定过表达目标基因尚未得到研究。在本研究中，我们验证了这种新的BSMV系统可以通过种子吸胀感染棉花，并在棉花中系统地过表达大片段基因（长达2340 bp）。与绿色荧光蛋白（GFP）融合的目标基因在棉花幼苗的根、茎和子叶中高水平表达，甚至在4周后仍能在棉花根和叶中检测到稳定的荧光信号。基于BSMV过表达系统，快速建立了定位于细胞核、内质网、质膜、高尔基体、线粒体、过氧化物酶体、液泡膜和质体的内源蛋白亚细胞定位标记系。利用BSMV系统过表达棉花胆汁酸钠转运体GhBASS5表明，GhBASS5通过将地下的Na转运到地上部分而负调控棉花的耐盐性。此外，多种蛋白质可以共同递送，通过共转化实现共定位和蛋白质-蛋白质相互作用的研究。我们还证实，通过递送分裂的SpCas9/sgRNA，BSMV系统可用于在棉花中进行无DNA基因编辑。最终，本研究表明，这种BSMV系统可作为未来棉花基因功能研究的高效过表达系统。
