Expression of human glucocerebrosidase gene in transgenic green algae Dunaliella salina Teod. / Poramate Klanrit = การแสดงออกของยีนกลูโคซีรีโบรซิเดสของคนในสาหร่ายสีเขียว Dunaliella salina Teod. ที่ได้รับการถ่ายยีน / ปรเมษฐ์ กลั่นฤทธิ์ f
Dunaliella salina Teod., a green microalgae and a source of B-carotene production, is a salt tolerant algae able to grow at high salt concentration and having potential for industrial production. The optimization of conditions for culturing of this algae using modified JR medium was investigated. Results revealed 0.25X JR at pH 8.5 was the most effective condition providing maximum growth. When this JR was compared with the original recommended J/1, results showed that cell proliferation in JR was found higher than those in J/1, supporting JR medium as a substitution. Genetic transformation in D. salina using bialaphos-resistant gene (bar) as a marker gene was carried out based on PEG-mediated transformation. Among 6 levels of concentrations investigated, 0%, 0.02%, 0.04%, 0.06%, 0.08% to 0.1% w/v, A 0.08% PEG revealed most effective in providing the highest number of cells resistant to bialaphos herbicide. Molecular analyses of DNA via PCR and RNA via RT-PCR were also revealed the integration of target DNA and expression of mRNA, respectively, as evidence by DNA bands similarly found in those of positive control. When Glucocerebrosidase gene (GBA) was employed to investigate the possibility of using D. salina as a model system to produce human recombinant protein using GBA gene connected with pBicBar with the condition previously obtained in bar gene transfer. The transformants presented with 5x10 4 cells/ml after 1 month in selective condition. Molecular analyses via PCR and RT-PCR revealed a positive results both GBA DNA insertion and mRNA expression, the transformation was successful and the expression of GBA in D. salina was occurred at RNA level. These results showed the possibilityh in using this expression system as a basis for future applications.
