| Dunaliella salina(D.salina) is a unicellular green alga without cell wall and one of the most halotolerant eukaryotes now known. The significant difference between D.salina and other green unicellular algae is that the cell of D.salina lacks a rigid cell wall, and is enclosed by a thin layer of elastic plasma membrane. D.salina has the shape of pear or ellipse with volume of 50-100uM3, The cell of D.salina has two long apical flagella that propel it through the water. In the cell, there is the single, large, cup-shaped chloroplast with a pyrenoid. The red eyespot is located at the front of the cell. Reproduction of D. salina includes a sexual reproduction or a longitudinal split as the main manner and sexual reproduction in isogancy. The extraordinary features of D.salina make it a model system to study tolerance to extreme environments at the molecular level. In addition, D.salina, beta-carotene-accumulating alga, has been cultivated in China and other countries as a source of retinol and beta-carotene. Due to its potential advantages over microbial, fungal system and transgenic animals, D.salina are attracting interest as bioreactors for the inexpensive production of recombinant proteins, such as vaccines and antibodies. In this study, we studied on biological behavior and selectable marker of D.salina to construct the expression vector pGEM-D 18S-BAR. Methods and results:1. Purification: centrifugal elutriation, repeated culture and cultured with chloromycetin were used to purify D.salina in liquid medium, then stranded on 0.8% agar plates.2. Quantification: three methods including wet weight, UV-visible absorption and counting were used to measure D.salina. By use of scanning at 600-800 nm, 685nm and 680nm were considered as absorption wave length of D.salina. When diluted with distilled water, the algal cells swelled quickly and became moveless, then they could be counted.The three methods could be used for quantification, and the relations between the methods were established.3. Selection of optimal medium: Through comparing cell number, growth rate, concentration of total protein and beta-carotene of D.salina, the optimal medium was available.4. Determination of selectable marker: D.salina exhibited sensitivities to chloromycetin and phosphinothricin. When the initial number of D.salina was 1-2X106/ml, effective concentration of 90%(EC9o), after 10 days of exposure to chloromycetin, ranged from 143.63 to 219.51mg/l, except D.salina 435 (414.62mg/l) . For phosphinothricin, EC9oafter 10 days of exposure to it ranged from 4.44 to 6.49mg/l. Effective concentrations which inhibited the growth of non-transgenic D.salina were primarily confirmed, doses of chloromycetin and phosphinothricin were 200mg/l and 3.0mg/l, respectively.5. Amplification and cloning of the 18S rDNA fragment of D.salian (UTEX LB 1644): Genenomic DNA of D.salina was used as a template, the PCR primers were designed according to the D.salina 18S rDNA gene sequence (Genbank access number M84320), subsequently, a 1122bp fragment was amplified. Then, the PCR product was cloned into vector pGEM-7zf to create a vector pGEM-S18S. After the fragment was sequenced, a BLAST search of nucleic acid sequences present in Genbank was made which revealed that 98%-99% of sequences of the fragment was homologous with the sequences of D. salina, suggesting that the fragment should be 18S rDNA fragment of D.salina.6. Construction of the expression vector pGEM-D18S-BAR: two copies of the 18S rDNA fragment of D.salina were ligated with pGEM-7zf to get a vector pGEM-D18S. Bar gene, encoded phosphinothricin acetyltransferase that detoxifies phosphinothricin, drived by cauliflower mosaic virus (CaMV) 35s promoter, and terminated by nopaline synthase (NOS) 3' terminator was obtained from plasmid pBARGUS by digestion. This fragment was cloned into vector pGEM-D18S to constrct expression vector pGEM-D18S-BAR.7. Transformation and selection: A gene gun method was employed for the transformation, then selected by phosphinothrici...
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