Construction of Novel Chloroplast Expression Vector and Development of an Efficient Transformation System for the Diatom Phaeodactylum tricornutum

• DOI: 10.1007/s10126-014-9570-3
• Publication Date: 2014-04-25T02:25:12Z
• Authors: Wei-Hong Xie; Cong-Cong Zhu; Nai-Sheng Zhang; Da-Wei Li; Wei-Dong Yang; Jie-Sheng Liu; Ramalingam Sathis...; Hong-Ye Li
• Keywords: Diatoms; 0301 basic medicine; 0303 health sciences; Chloroplasts; Microscopy, Confocal; Blotting, Western; Genetic Vectors; Green Fluorescent Proteins; Chromosome Mapping; Drug Resistance, Microbial; Enzyme-Linked Immunosorbent Assay; Aquatic Science; Flow Cytometry; Real-Time Polymerase Chain Reaction; Applied Microbiology and Biotechnology; 03 medical and health sciences; Chloramphenicol; Electroporation; Transformation, Genetic; Genes, Reporter; Original Article; Cloning, Molecular; Biotechnology; DNA Primers

Plastids are ideal subcellular hosts for the expression of transgenes and have been successfully used for the production of different biopolymers, therapeutic proteins and industrial enzymes. Phaeodactylum tricornutum is a widely used aquatic feed species. In this study, we focused on developing a high-efficiency plastid expression system for P. tricornutum. In the plastid transformation vector, the site selected for integration was the transcriptionally active intergenic region present between the trnI and trnA genes, located in the IR (inverted repeat) regions of the plastid genome. Initially, a CAT reporter gene (encoding chloramphenicol acetyltransferase) was integrated at this site in the plastid genome. The expression of CAT in the transformed microalgae conferred resistance to the antibiotic chloramphenicol, which enabled growth in the selection media. Overall, the plastid transformation efficiency was found to be approximately one transplastomic colony per 1,000 microalgae cells. Subsequently, a heterologous gene expression cassette for high-level expression of the target gene was created and cloned between the homologous recombination elements. A TA cloning strategy based on the designed XcmI-XcmI sites could conveniently clone the heterologous gene. An eGFP (green fluorescent protein) reporter gene was used to test the expression level in the plastid system. The relatively high-level expression of eGFP without codon optimisation in stably transformed microalgae was determined to account for 0.12 % of the total soluble protein. Thus, this study presents the first and convenient plastid gene expression system for diatoms and represents an interesting tool to study diatom plastids.