Construction of novel chloroplast expression vector and development of an efficient transformation system for the diatom Phaeodactylum tricornutum.
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.
Key Laboratory of Eutrophication and Red Tide Prevention of Guangdong Higher Education Institute, Jinan University, 510632, Guangzhou, China.
Wei-Hong
WH
Xie
Cong-Cong
CC
Zhu
Nai-Sheng
NS
Zhang
Da-Wei
DW
Li
Wei-Dong
WD
Yang
Jie-Sheng
JS
Liu
Ramalingam
R
Sathishkumar
Hong-Ye
HY
Li
D015153:Blotting, Western; D002701:Chloramphenicol; D002736:Chloroplasts / Q000235:genetics*; D002874:Chromosome Mapping; D003001:Cloning, Molecular; D017931:DNA Primers / Q000235:genetics; D017377:Diatoms / Q000235:genetics*; D004352:Drug Resistance, Microbial / Q000235:genetics; D018274:Electroporation; D004797:Enzyme-Linked Immunosorbent Assay; D005434:Flow Cytometry; D017930:Genes, Reporter / Q000235:genetics; D005822:Genetic Vectors / Q000235:genetics*; D049452:Green Fluorescent Proteins / Q000235:genetics; D018613:Microscopy, Confocal; D060888:Real-Time Polymerase Chain Reaction; D014170:Transformation, Genetic / Q000235:genetics*
D017931:DNA Primers; D049452:Green Fluorescent Proteins; D002701:Chloramphenicol