Abstract
Tetraselmis subcordiformis, a unicellular marine green alga, is used widely in aquaculture as an initial feeding for fish, bivalve mollusks, penaeid shrimp larvae, and rotifers because of its rich content of amino acids and fatty acids. A stable nuclear transformation system using the herbicide phosphinothricin (PPT) as a selective reagent was established previously. In this research, the recombinant expression in T. subcordiformis was investigated by particle bombardment with the rt-PA gene that encodes the recombinant human tissue-type plasminogen activator (Reteplase), which is a thrombolytic agent for acute myocardial infarction treatment. Transgenic algal strains were selected by their resistance to PPT, and expression of rt-PA was validated by PCR, Southern blotting, and Western blotting, and bioactivity of rt-PA was confirmed by the fibrin agarose plate assay for bioactivity. The results showed that rt-PA was integrated into the genome of T. subcordiformis, and the expression product was bioactive, indicating proper post-transcriptional modification of rt-PA in T. subcordiformis. This report contributes to efforts that take advantage of marine microalgae as cell factories to prepare recombinant drugs and in establishing a characteristic pathway of oral administration in aquaculture.
Highlights
Tetraselmis subcordiformis (Wille) Butcher (= Platymonas subcordiformis) is a common marine unicellular green alga with broad application prospects in aquaculture and bioenergy [1,2,3,4]
Due to its high quantities of polysaccharides, proteins, polyunsaturated fatty acids, vitamins, and other nutrient supplements [4,5,6,7], T. subcordiformis is widely used in aquaculture as feed for bivalve mollusks, penaeid shrimp larvae, and rotifers [2,8,9]
T. subcordiformis was shown to photobiologically evolve hydrogen (H2), indicating that this alga may provide a potential sustainable energy source [3]. Both stable nuclear and chloroplast transformation systems have been established in T. subcordiformis using glass-bead agitation and the particle bombardment method [10,11]
Summary
Tetraselmis subcordiformis (Wille) Butcher (= Platymonas subcordiformis) is a common marine unicellular green alga with broad application prospects in aquaculture and bioenergy [1,2,3,4]. T. subcordiformis was shown to photobiologically evolve hydrogen (H2), indicating that this alga may provide a potential sustainable energy source [3]. In earlier research, both stable nuclear and chloroplast transformation systems have been established in T. subcordiformis using glass-bead agitation and the particle bombardment method [10,11]. Following biolistic transformatiioonn and recovery for two days, all bboommbarded cceelllls were ttrraannssffeerrrreeddttoolliiqquuididmmooddifiifeieddf/f/22mmeeddiiuummwwiitthh5500mmgg·L·L−−11 ooff PPPPTT and cultured for a week. During this selection process, most algal cells died and the color of medium turnedd from green to white. TSaabmSlpealme1s. pQlBeuisoamntaistas t(BiFvrieeosmdWheaeWtseisgermih(gFthi)rnte)asthiSoonluobSf loreelPucrobomlteebiPninroantrett-iPrntA-PA in transgenic samplCeso.ncentration rt-PA
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