Comprehensive plasmid toolkit for multipart assembly in the Dunaliella salina nuclear system

Dunaliella salina is a kind of green algae with strong salt tolerance and stable genetic characteristics. However, at present, the current research is not convenient and rapid to fine-tune the multi-gene expression of D. salina. In this study, a plasmid toolkit for expression in the nuclear system of D. salina was designed and constructed by using the top-down hierarchical construction method and Golden Gate technique, each biological element could be replaced by an IIS restriction endonuclease site (BsmBI or BsaI), the toolkit was able to achieve the expression of multiple genes in the nuclear system of D. salina in a short time. The laser confocal images showed that the multi-gene plasmids of the tool kit could be successfully transferred into D. salina cells, and the promoters and terminators could work normally, and the gene could be successfully expressed. The results of real-time quantitative PCR showed that the selected promoters of the tool kit had various expression intensities, which could make it meet the different expression needs of the experiment. •A novel plasmid toolkit has been designed for multi-gene expression in the nuclear system of D. salina•The toolkit enables modular substitution of biological components for precise multi-gene expression.•The transformation efficiency of toolkit in the absence of screening for resistance is in the range of 1.69 to 3.48%.•The initiation strengths of the promoters were ranked in descending order as follows: rbcs, lcyb, ip, mray, bkt.