[Construction of Dunaliella salina heterotrophic expression vectors and identification of heterotrophically transformed algal strains].
We constructed inducible and constitutive heterotrophic expression vectors of Dunaliella salina (D. salina) and identified heterotrophic transformants. A gene encoding a glucose transporter (Glut1) was cloned from human placenta tissues by RT-PCR and sequenced. Inducible heterotrophic expression vector pMDDGN-Bar of D. salina, which included a duplicated carbonic anhydrase (DCA) promotor and a Bar selectable marker that could drive expression of the Glut1 gene in D. salina, was constructed by molecular biology methods. In addition, we constructed another vector G5Glut1-Bar that contained a constitutive ubiquitin promotor, Glut1 and Bar box. The two expression vectors were introduced into D. salina by electroporation method, and then screened the transformants with phosphinothricin (PPT). Total RNA of the transformants extracted was used to analyze the integration of the target gene (Glut1) by RT-PCR. The cloned Glut1 sequence was 1479 bp and encoded 493 amino acids. The results of all enzymes digesting showed that two expression vectors were successfully constructed. After screening by PPT for several weeks, the transfomants grew well whereas wild-type cells died completely. The result of RT-PCR indicated that two transformants both had an about 250 bp specific band and the sequence homology was 100% compared with the human Glut1 sequence by Blast analysis. Taken altogether, inducible and constitutive heterotrophic expression vectors of D. salina was constructed successfully and the Glut1 gene was integrated into the genome of D. salina. Expression vectors above-mentioned may be used for the expression of the foreign Glut1 gene in D. salina.
Chen
Tao
T
Laboratory for Cell Biology, Department of Bioengineering, Zhengzhou University, Zhengzhou 450052, China.
Liu
Hongtao
H
Lü
Pengju
P
Xue
Lexun
L
D001483:Base Sequence; D000460:Chlorophyta / Q000235:genetics / Q000378:metabolism*; D003001:Cloning, Molecular; D018274:Electroporation; D005822:Genetic Vectors / Q000235:genetics*; D051272:Glucose Transporter Type 1 / Q000096:biosynthesis / Q000235:genetics*; D007218:Industrial Microbiology; D008969:Molecular Sequence Data; D014170:Transformation, Genetic
D051272:Glucose Transporter Type 1