CONSTRUCTION OF AN EGFP EUKARYOTIC EXPRESSION VECTOR BY SOEING AND ITS TRANSFORMATION TO DUNALIELLA SALINA

Previous studies,including some from our own laboratory,have demonstrated that Dunaliella salina (D. salina) was one of the most extremely halotolerant eukaryotes and able to live in a variety of salt concentrations ranging from 01 05 to 5M solu2tion of sodium chloride1The simple and cheap culture of D. salina makes it have a great potential in bioengineering for producingvaluable polypeptides and proteins1However, lack of efficient expression systems has been one of major limitations in the geneticmanipulation of this microalga1In order to produce high levels of heterologous proteins in transgenic D. salina,it is necessary toobtaina high2efficiently endogenous promoter for expressions of the heterologous genes under controlled conditions1We investi2gated whether 5′2flanking region (Pnr) and 3′2flanking region (Tnr) of D. salina nitrate reductase (NR) would control expres2sion of the heterologous genes1Gene splicing by overlap extension (SOEing) was modified according to following steps: (1) syn2thesis of two individual DNA fragments of interest with 36 bp overlap by PCR with high2fidelity Pyrobest DNA polymerase ;(2) six cycles of pre2extension without flanking primers after mixing the two fragments above, which ensures the overlap exten2sion between the two individual templates ; (3) 25 cycles of post2extensionwith flanking primers after cooling the product of pre2extension1An enhanced green fluorescence protein(EGFP) eukaryotic expression vector harboring Pnr2Tnr cassette was construct2ed based on the method of SOEing and transformed into D. salina using electroporation1Using the modified SOEing PCR, a spe2cial fusion fragment Pnr2EGFP was obtained with the equal molar Pnr and EGFP cDNA from the first stage PCRs being templates,and a recombinant expression vector p7NET containing Pnr2EGFP2Tnr expression cassette was constructed by inserting Pnr2EGFPand Tnr, which was obtained from the genome of D. salina by PCR, into vector pEGm27zf1The resulting recombinant vectorp7NET was digested with the double enzymes AatⅡand XhoⅠand a single enzyme HindⅢ, which showed the expected frag2ments, respectively1The results of sequencing showed that the sequences of whole Pnr2EGFP2Tnr expression cassette completely conformed to the corresponding sequences of Pnr, EGFP and Tnr, which provides a further evidence for successful construction ofthe recombinant plasmid by SOEingSubsequently, the transformants of D. salina transformed with the p7NET were observed under fluorescence microscope. Green fluorescence appeared on day 2 after transformation in D. salina cells transformed with the p7NET, but not in control cells,in which the chloroplast red autofluorescence appeared. The transformed cells on day 6 after transformation had stronger green fluorescence than those on day 2, but no noticeable fluorescence on day 9,indicating that the EGFP gene has successfully been introduced to cells of D. salina and Pnr from D. salina can drive the expression of the EGFP gene in transgenic D. salina. Conclusions: The findings of the present study suggest that the Pnr/ Tnr cassette of the NR gene from D. salina may be used to drive the expressions of heterologous gene in transgenic D. salina and the modified SOEing is a rapid and efficient method in cloning of fusion fragments and construction of expression vectors.