Abstract
The objective of this research was to establish a chloroplast transformation technique for Platymonas (Tetraselmis) subcordiformis. Employing the gfp gene as a reporter and the bar gene as a selectable marker, transformation vectors of P. subcordiformis chloroplast were constructed with endogenous fragments rrn16S–trnI (left) and trnA–rrn23S (right) as a recombination site of the chloroplast genome. The plasmids were transferred into P. subcordiformis via particle bombardment. Confocal laser scanning microscopy indicated that the green fluorescence protein was localized in the chloroplast of P. subcordiformis, confirming the activity of the Chlamydomonas reinhardtii promoter. Cells transformed with the bar gene were selected using the herbicide Basta. Resistant colonies were analyzed by PCR and Southern blotting, and the results indicated that the bar gene was successfully integrated into the chloroplast genome via homologous recombination. The technique will improve genetic engineering of this alga.
Highlights
Platymonas (Tetraselmis) subcordiformis is a marine unicellular green alga that is a widely used feed in aquaculture for its high nutrient levels
In the plasmid pPSCB, the bar gene was used as the selectable marker to confer algal resistance to the herbicide Basta, while the plasmid pPSC–GFP used the gfp gene as the reporter
The bar and gfp genes were each driven by the C. reinhardtii chloroplast regulators 59 untranslated region (UTR) of atpA and 39 UTR of rbcL
Summary
Platymonas (Tetraselmis) subcordiformis is a marine unicellular green alga that is a widely used feed in aquaculture for its high nutrient levels. Chloroplast transformation has many advantages over nuclear transformation, such as the high expression level of foreign genes [2], the homologous integration of foreign genes [3], the lack of gene silencing and position effects [4], and co-expression of multigenes [5]. For P. subcordiformis, chloroplast transformation could be more suitable than nuclear transformation to raise its nutrient value. A strategy to increase hydrogen production is to regulate PS I and PS II to maintain cells anaerobic by manipulating genes encoding PS proteins via chloroplast transformation. The current lack of chloroplast transformation protocols is obstructing this promising and fundamental research
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