Construction of Novel Chloroplast Expression Vector and Development of an Efficient Transformation System for the Diatom Phaeodactylum tricornutum
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
DOI:
10.1007/s10126-014-9570-3
Publication Date:
2014-04-25T02:25:12Z
AUTHORS (8)
ABSTRACT
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.
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