Establishment Of The Nuclear And Chloroplast Transformation System Of Platymonas Subcordiformis

Platymonas (Tetraselmis) subcordiformis (Prasinophyceae, Chlorophyta) is a unicellular marine green alga with great value in aquaculture and new engineering. The basic research and wild amplification of P. subcordiformis were hindered by the lack of a stable transformation system. This article is to establish the nuclear and chloroplast transformation system of P. subcordiformis, to improve the fundamental research and enlarge application of this alga.P. subcordiformis was found to be very sensitive to the herbicide Basta through sensitivity test, therefore, the herbicide Basta could be employed as a selective agent, and the bar gene is a practicable selectable marker gene.Stable nuclear transformation system was established by pariticle bombardment and glass-bead agitation. The method of glass-bead agitation is only available for protoplasts. Therefore P. subcordiformis was firstly incubation with abalone acetone powder to produce protoplasts. Then the plasmid pEGFP-N1 was transferred into the protoplasts by glass-bead agitation, and the transient expression of GFP was observed. The result probed that the glass-bead agitation method is applicable for P. subcordiformis. Following that, the vector containing expression cassette of bar gene was transferred to P. subcordiformis by particle bombardment and glass-bead agitation. Then the transformed cells were selected by Basta. At last the results of Southern blotting analysis indicated that the bar gene was successfully integrated into nuclear genome of P. subcordiformis using both the two transgenic techniques. The transformation efficiency of galss-bead agitation is higher than that of particle bombardment.Stable chloroplast transformation system was established by particle bombardment. Transformation vectors for P. subcordiformis chloroplast were constructed with rrn16S-trnI (left) and trnA-rrn23S (right) of the IR region as a recombination site. The plasmid was constructed with the 5'atpA and 3'rbcL from Chlamydomonas reinhardtii as regulators, and the vector pPSCG was inserted with the gfp gene as a repoter while pPSCB inserted with the bar gene as a selectable marker. Plasmids were transferred into P. subcordiformis via particle gun mediated transformation.Cells transformed with the gfp gene were selected by flow cytometry, and through the test of subcellular localization by laser scanning confocal microscope, GFP was localized in chloroplast. These results indicated that the particle gun method and the regulators were fit for the chloroplast transformation of P. subcordiformis.Cells transformed with the bar gene were selected by herbicide Basta. The results of PCR and Southern blotting indicated that the bar gene was successfully integrated into the chloroplast genome via homologous recombination in transformants.