| Dunaliella salina (D. salina) is one of the most halotolerant unicellular eukaryote known and it can live in a variety of salt concentrations ranging from 0.05 to 5M solution of sodium chloride. Therefore, the ease and cost-effectiveness of culturing D. salina make it a desirable target for increased production of natural compounds by genetic engineering or for exploitation as biological factories for the synthesis of novel high-value compounds. However, the lack of efficient genetic transformation systems has been one of major limitations in the manipulation of this microalga.The 70 KDa heat shock protein (hsp70) is one of the most highly conserved hsps. In vascular plants and green algae, various hsp70 species are present in the cytosol, mitochondria, endoplasmic reticulum and chloroplast, which are reqired for the stabilization of the partially unfolded translation-competent form of polypeptides and are released from the polypeptides upon hydrolysis of ATP. The induction of light and heat stresses make a significantly transient accumulation of cytosoli-localized hsp70 transcripts. The hsp70 promoter, therefore, has been successfully used to construct the expression vector in chlamydomonas. In this study, the cytosoli-localized hsp70 DNA from D.Salina was isolated and the correlation between the mRNA level of hsp70 and temperature and light was also investigated. The hsp70 promoter of D.Salina has been proposed to be useful element for the vector construction of transgenic D.Salina.Some reliable transformation selection protocols as well as appropriate promoters that work in D. salina are prerequisites for the development of a practical transformation system. Antibiotic resistance genes have been used as selectable markers in C. reinhardtii and Volvox, but expression of antibiotic resistance genes often failed due to inappropriate codon usage. Moreover, D. salina was naturally resistant to most of antibiotics such as spectinomycin, kanamycin or chloramphenicolcommonly used for selection of genetically transformed plant cells, presenting further difficulty in developing selection strategies for recovery of the transformed cells of D. salina. In contrast, D. salina cells were strongly sensitive to phosphinothricin (PPT). The bialaphos resistance gene (bar) from streptomyces hygroscopicus encodes a phosphinothricin acetyl transferase (PAT) that detoxifies PPT by catalyzing the addition of an acetyl group to the amino group of PPT. Since D'Halluin at al used the bar gene as a selectable marker in higher plant engineering, it has been widely exploited in transgenic plants. Selection for bar gene activity can be achieved easily and inexpensively, and controlled effectively in plant engineering with the bar gene. So far, however, successful selection for transformants of D. salina harboring the bar gene and resistant to PPT has not been reported. In order to drive high levels of foreign gene expression, native promoters highly active in D. salina are necessary. In the present study, we isolated and characterized the 5' regulatory regions of the highly expressed actin gene of D. salina, and constructed a D. salina transformation cassette containing the actin promoter which was able to drive expression of the bar gene, showing that the bar gene can serve as a dominant selectable marker in this alga.Part one: Cloning and characterization of a cytosoli-localized hsp70 DNA from D. salina Methods1. Two pairs of degenerate primers were designed according to the conserved region of cytosoli-hsp70 amino acid sequence from various species, and used to amplify cDNA fragment from heat-shock-treated D. salina by nest PCR. The resulting PCR products were inserted into T-vector and then sequenced.2. RACE protocol was performed to obtain the full-length cDNA, 5'UTR and 3'UTR of the hsp70.3. The genomic DNA from Dunaliella salina was digested with Dra I, EcoR V, Pvu II and Stu I, respectively. A Genome Walker Adaptor was ligated to the ends of the digested DNA fragments. Genome Walker Libraries, including GWL1, GWL2, GWL...
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