Transformation of Dunaliella salina by using glass beads--a novel transformation method / 生物工程学报

A novel transformation method was firstly established using glass beads in Dunaliella salina (D. salina). The results showed that the GUS gene, a reporter gene, was successfully expressed in D. salina. Cells of D. salina presented blue color under the microscope after stained. In addition, different factors which influenced transformation were optimized including the transformation consecutive time, rotate speed, concentration of the plasmid and PEG 6000. The experiment indicated that this fit together can obtain the best results for D. salina transformation: adding 150 microL PEG and 90 microL plasmid DNA to 800 microL culture of D. salina (10(6) cells/mL) containing 300 mg glass beads, swirling 12 seconds under the rotate speed 2400r/min. This newly method can be used as a potential tool in the research of D. salina gene engineering with the advantage of more simpleness, convenience, quickness and less expense.

Regulation of a Highly Repeated GT Sequence from DCA1 Promoter of Dunaliella Salina in Salt-induction / 中国生物工程杂志

To identify whether a highly repeated GT sequence from DCA1 promoter from Dunaliella salina,which have been proved to be a salt-inducible promoter in our previous study,would be a salt-inducible regulation element,different primers were designed to amplify 6 different-length fragments of DCA1 promoter from D.salina by PCR.After these fragments were respectively inserted into the HindⅢ-BamH I sites of the vector pU?GUS,serial expression vectors containing the gus gene were generated.D.salina cells transformed with these recombinant plasmids by electroporation were grown in liquid media containing different concentrations of sodium chloride respectively.GUS enzyme activity was measured histochemically and fluorometrically.The results revealed that 3 fragments containing GT repeated sequence drove the external gus gene expression and the expression pattern of the gus gene was regulated by the concentrations of sodium chloride.Additionally,the 2 fragments without tandem GT sequence drove the gus gene expression,but the expression pattern of the gus gene wasn't regulated by the concentration of sodium chloride;Also,the upstream fragment of the tandem GT sequence wasn't able to drive the gus gene expression.In conclusion,the highly repeated GT sequence from the DCA1 promoter plays an important role in the salt-inducible regulation of DCA1 promoter from D.salina and might be a novel salt-inducible element.

Preliminary Study on Transformation of Human canstatin Gene to the Novel Bioreactor——Dunaliella salina / 中国生物工程杂志

The human canstatin cDNA was amplified by RT-PCR and then directionally cloned into pU? expression vector. The recombinant pU?-Can vector was connected with the screening marker (bar box), to construct a eukaryotic expression vector called pU?-Can-Bar. This expression vector was introduced into the D.salina by glass beads method. The screening culture of transformants of D.salina was performed in solid media containing 5 ?g/ml PPT, and the analyses of transformants were carried out through PCR and Southern blot. PCR results revealed that specific 700 bp products were detected in the different transformants of D.salina but not in negative control. Southern blot analysis further demonstrated that human canstatin gene was integrated into the D.salina genome. Moreover, the results of genetic stability analyses of transformants demonstrated that canstatin gene was stably inherited in the D.salina transformants. The successful preparation of the D.salina transformants will provide the experimentation evidence for producing canstatin protein cosmically by using the D.salina bioreactor and give a better prophase work basis for clinic application of canstatin protein early.

Prospect and Progress on Dunaliella salina in the Area of Molecular Biology / 中国生物工程杂志

Unicellular green alga,Dunaliella salina(D.salina),is a biflagellar alga without cell wall,which is a kind of very important eukaryotic microalga.In the previous study,the research of D.salina focus on the morphology,the mechanism of salt tolerance and ?-carotene,however,with the rapid development of microalgal biotechnology,a lot of work about D.salina was reported in recent years.In the area of molecular biology,the studies of D.salina mainly place emphasis on the cloning and analysis of important functional genes,regulatory sequences,and the expression of foreign genes using D.salina as host.The research advance in these aspects were reviewed.

Comparison of Stable Expressions of Foreign Genes Driven by Different Promoters in Transgenic Dunaliella salina / 中国生物工程杂志

The purpose was to compare the difference between transgene expressions driven by homologous duplicated carbonic anhydrase (DCA) promoter and foreign CaMV35S promoter in the unicellular green alga, Dunaliella salina(D.salina).The CaMV35S promoter-bar construct and DCA promoter-bar construct into D.salina by a Backon 2000 electroporation system were introduced. After the repeated selections with the phosphinothricin (PPT) of 3mg/L, 3 PPT-resistant phenotype transformants were isolated from the CaMV-bar and DCA-bar pools of transformants of D. salina, respectively. The results of PCR and sequencing showed that bar genes were stably integrated into the genome of D.salina, and Southern bolts showed the number of transgene copy had no significant difference between both promoters. Semi-quantitive RT-PCR indicated that the mRNA levels of bar gene were higher in DCA-bar transformants than the CaMV-bar transformants, and could be increased under the induction of high salt in DCA-bar transformants but not in the CaMV-bar transformants. Analysis of growth rate of transformants showed DCA-bar transformants achieved the log stage faster than the CaMV-bar transformants. It is concluded that the homologous promoters have more advantages than the foreign promoters in the transgenic D.salina.

Cloning and Characterization of 5′Flanking Region of Nitrate Reductase Gene Derived from Dunaliella salina / 中国生物工程杂志

Aim: Clone and characterize of the 5′- flanking region of the nitrate reductase (NR) gene derived from Dunaliella salina(D. salina). Methods : The genomic DNA from D. salina was respectively digested with BamHI, EcoRI, HindIII, Pst I, Sal I and Xba I. A genomic walking cassette was ligated to the ends of the digested DNA fragments, and then genomic walking libraries comprising BL, EL, HL, PL, SL and XL were constsucted. The 5′- flanking region of the NR gene from genomic walking libraries of D. salina was amplified by LA-PCR. The DNA sequences were analyzed with the software - Promoter Predictions. Isolated 5′-flanking regions fused to the GUS gene were tested for transient expression in the alga. Results: A single specific PCR product of about 1200bp in length from the HL library was generated. Also, several conserved motifs, such as CAAT-box, GAGA-box were found, which are related to regulation of transcription, and the putative binding sites of transcriptional factors such as EBP, EFII, NF-E1 and LV. BLAST showed that the DNA sequences shared high homology with 5′-upstream region of the NR gene from Dunaliella viridis. The isolated 5′-flanking regions were able to strongly drive GUS reporter gene expression, suggesting that it contains the promoter elements necessary for the transcription of the NR gene. The expression pattern of the GUS gene and the NR gene were similar, both ware induced by nitrate and repressed by ammonium. Conclusion: The cloned 5′- flanking sequences of NR gene derived from D. salina might be a specific promoter with the ability to“switch on or off” an expression of the heterologous gene in transgenic D. salina.

Cloning and analysis of psaB cDNA of Dunaliella salina / 生物工程学报

One pair of degenerate primer was designed according to conserved motifs of the psaB (A2 subunit of photosystem I) of Chlamydomonas reinhardtii, Chlamydomonas moewusii, Chlorella vulgaris and Mesostigma viride, and a total RNA of Dunaliella salina (D. salina) was extracted with TRIzol reagent. A cDNA fragment, about 1.8kb in length, from green algal D. salina was obtained through RT-PCR method. The resulting PCR product was cloned into T-vector and screened to determine its sequence. Homologous analysis of the deduced amino acid sequence was performed by BLAST and subsequeqtly compared with GenBank data. The obtained cDNA sequence was 1815 bp long, which encodes 605 amino acids (GenBank accession number: AY820754). The sequence shared high homologue with the following psaB: Chlamydomonas reinhardtii 92%, Chlamydomonas moewusii 91%, Chlorella vulgaris 86%, Mesostigma viride 85%, Physcomitrella patens subsp. Patens 85% and Nephroselmis olivacea 84%. It can be concluded that the cloned sequence is psaB cDNA fragment from D. salina.