Construction Of A Light Inducible Expression Vector And Its Application In Transformation Of Dunaliella Bardawil

The alga Dunaliella, such as Dunaliella salina and Dunaliella bardawil, is an autotrophic unicellular eukaryotic green alga which looks much like another much related unicellular eukaryotic green alga, Chlamydomonas reiharditti, in morphology. But the varieties of Dunaliella species differ from C. reiharditti in two aspects of biological characteristics; they are halotolerant and lack a rigid cell wall. Dunaliella algae potentially are ideal candidate hosts for bioreactor, which can be used to generate many transgenic products including many pharmaceutical proteins, such as the interferins, interleukin-2 (IL-2), and tumor necrosis factor (TNF), etc. Dunaliella, as a bioreactor host, has a great deal of advantages over other model organisms. Since Dunaliella belongs to autotrophic organism, the culture of Dunaliella needs only light, water, and some inorganic salts without the use of costly organic medium as in the culture of E.coli and yeast, that is, the cost of Dunaliella is relatively very low. Dunaliella cells are much halotolerant, they can propagate in medium of 0.05-5.0 mol/L sodium chloride, and the medium most often used in laboratory contains 1.0-2.0 mol/L sodium chloride which prevents propagation of other microorganisms, implying that the laboratorial culture of Dunaliella is easier and could efficiently avoid the contamination of other microbes. Since Dunaliella cell has no cell wall, it is convenient to make geneticmanipulation on it, i.e. it is facile to introduce foreign DNA into Dunaliella cells, and even, many procedures of transfection of mammalian cells, as electroporation, phosphate calcium coprecipation, and liposome-mediated transfection, can be assumed to transform Dunaliella. Besides, on transgenic consideration, since the genome of vegetative Dunaliella cell is haploid, the expression of transgene is direct and manifest without the interference of recessive effect as in diploid cells, and this makes the screening of Dunaliella mutant facilitated. Finally, because Dunaliella cells are eukaryotes, the proteins inside them can be processed after translation, accordingly, the protein product by Dunaliella genetic engineering behaves more like the mammalian protein than that of E.coli gene engineering, namely, protein product from Dunaliella genetic engineering, in industry, requires less posttranslational procession than that from prokaryotic organisms. Another advantage for protein manufactured from Dunaliella is that we can use Dunaliella to produce edible vaccine for Dunaliella itself is safe to human being. In comparison of tansgenic higher plants, Dunaliella cells propagate rapidly and independent of seasons guaranteeing the production of protein at any moment in a year.But most studies on Dunaliella, so far, have been focusing on the mechanism of its halotolerance and on production of carotene, and few transgenic studies are performed on Dunaliella. The purpose of this study is to construct an expression vector and to find some methods that can be used to transform Dunaliella bardawil, and finally to establish a bioreactor on the ground of Dunaliella.To begin the transgenic research of Dunaliella, the author designed and cloned a light inducible expression vector which is applicable totransiently and/or stably transform Duiialiella bardawil. In the vector mentioned above, the exogenous herbicide-resistant gene bar (Basta resistant) was put under control of the promoter of gene cbr (carotene biosynthesis related) from Dunaliella bardawil, and at the downstream of bar gene lay the 3-UTR (3'-untranslated region) of cbr acting as a terminator of gene expression. These three fragments comprised an expression cassette of bar which was expected to confer Dunaliella bardawil a resistance of phosphinothricin (the major ingredient of Basta). Meanwhile, the two fragments flanking bar may act as homologous fragments and make the bar gene integrated into the genome of Dunaliella bardawil by double cross-over. The homologous integration of bar into genome of Dunaliella bardawil would...