| Dunaliella salina,belonging to Volvocales, a kind of green unicellular algae with no cell wall. As it can grow in very large salinity range, from 0.05mol/L to 5.5mol/L, Dunaliella salina is considered to be a miracle of salt-resistant in autotrophic eukaryotic organisms. With the simple cell structure, it becomes a model extremophilic alga to research salt-tolerant mechanism and search for salt resistant genes. Recently, Dunaliella salina as a source of lipids and subsequent biodiesel production, which can also fix atmospheric carbon dioxide and reduce global warming, has become research focus. At present,the Dunaliella salina genomic project has not finished yet, there are only about 3,936 ESTs and 213 nucleotide in the Genbank of NCBI. Therefore, the gene message is not enough for the further research. However,cDNA library is an efficient method to screen and analyze valuable genes.Based on successful culturing of Dunaliella salina in the laboratory, the cell growth curve at 1.5 mol/L NaCl salinity was studied. High quality cDNA Library from Dunaliella salina cultured at 1.5mol/L NaCl was constructed by using Takara cDNA Library Construction Kit. The cDNA was synthesized by M-MLV with purified mRNA, after blunting the cDNA termini, EcoR I Adapters were ligated, digested by Not I, removed the short fragment by passing spin column, then the cDNA was ligated with pAP3neo vector, and electroporated the Escherichia coli DH10B Strain. The titer of primary cDNA library was 1.2×106cfu/mL, and the rate of recombinant was above 95%. The colony PCR results showed that the lengths of most inserts were larger than 500bp. By searching the NCBI Nucleotide collection (nr/nt) and Non human,Non mouse ESTs databases, 13 of 66 cloning sequences, highly homologous to the Dunaliella salina EST in NCBI Genbank. And the novel ESTs, probably encode novel genes. These novel ESTs laid the foundation for further cloning of the salt tolerance genes and added more genetic information for Dunaliella salina. |
PDF Full Text Request