| Dunaliella salina belongs to chlorophyta, Chlorophycease, Volvocales and its shape and structure are very similar to Chlamydomonas reinhardtii except for lacking of cell wall. It is a kind of unicellular alga, can swim through its diflagella and has a large cup-shaped chloroplast covering about 48% of the cell volume.D.salina can grow in extreme environment such as in a variety of salt concentrations ranging from 0.05 to 5M, and the optimum salt cencentration for growth is 2 to 3M. Because its growth condition is very simple, D.salina is a favorable host for producing pharmaceutical proteins.Nucleic transformation in plants has many problems such as the complicated manipulations and low efficiency expression of the foreign genes, but the chloroplast transformation may resolve the problems mentioned above. The chloroplast genome is very small and genetical manipulation in it is easier, so the foreign gene is sited-direct integrated into the chloroplast genome through homologous recombination, which is helpful to locating the foreign gene introduced at a certain site where it can be expressed efficiently. Furthermore, because the chloroplast DNA has many copies, it is very possible to express high the foreign genes in the chloroplast.In order to find a chloroplast transformation system of D. salina, we constructed a high efficiency expression vector, and the key to it is that wemust find out a powerful promoter for the vector. It has been shown that the atpA gene cluster in the C. reinhardtii chloroplast genome comprises the aptA, Psbl, cemA and atpH genes, which encode a a -subunit of the coupling-factor-I (CFi) ATP synthase, a small photo system II polypeptide, a chloroplast envelope membrance protein, and subunit III of the CFo ATP synthase, respectively. The four genes have a common functional promoter that lies immediately the upstream of psbl and the atpAgene.D. salina has high homologys to C.reinhardtti, so the powerful promoter may be evolved either in D. slina or in C. reinhartii. For finding this powerful promoter, we designed a pair of degenerate primers and amplified the atpA gene fragment from the chloroplast of D. salina by PCR according to conserved motifs of the homologous amino acid sequences of five kinds of algae. In this study, the strong promoter was cloned to a construct high efficiency expression vector of the chloroplast of D.salina. On the other hand, the fragment of the atpA gene directly from the chloroplast genome was cloned by PCR technique through designing degenerate primer, which provides a fast and simple method for cloning some genes from the chloroplast.Methods:To find the conserved motifs, known amino acid sequences were compared. According to the conserved motifs of the homologous amino acid sequences of about ten kinds of algae, we designed a pair of degenerate primer and amplified the atpA gene fragment from the chloroplast of D. salina by PCR technique. The resulting PCR products were inserted into pMD-18 T-vector, and then transformed into E.coli JM109. Positive colonies were selected to determine their sequences. Homologous analysis of the deduced amino acid sequences was performed by BLAST and subsequently compared with GenBank data. The cloned fragment was used as a probe, which was hybridized with the chloroplast DNA of D. salina by SouthernResults and discussion:1. The products of PCRThe products amplified by PCR were analyzed by electrophorisis and a bright brand of about 400bp appeared.2. Cloning of the atpA gene and identification of the recombinant plasmid.The resulting PCR products were inserted into PMD-18 T-Vector, and then transformed into E.coli JM109. Positive colones were selected to determine their sequences. By the methods of enzyme digestion and electrophorisis, there were the expected fragments in four colones, respectively.3. analysis the result of sequencing.Homologous analysis of the deduced amino acid sequence were performed by BLAST and subsequently compared with GenBank data. Finally, we got a 405bp nucleotide seque...
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