| The transgenic plants have been studying for more than ten years, which have some advantages including that the plants are autotrophy not requiring expensive media or strict culture conditions, and the plant viruses cannot infect human, compared with other systems such as microbe fermentation and transgenic animals. But because the higher plant growth is so slow and restricted by seasons, interests of the studies are focused on the unicellular eukaryotic algae.Dunaliella salina (D. salina) belongs to Chlorophyta, Chlorophyceae, Volvocales and its shape and structure are very similar to Chlamydomonas reinhardtii (C. reinhardtii) except for lacking of cell wall. It is a kind of ellipse or pear-shaped unicellular tolerant alga with the length of six to fifteen um. It can swim through its diflagella and has a large cup-shaped chloroplast which comprises about 48% of the cell volume. D.salina can grow in extreme environment such as in a variety of salt concentrations ranging from 0.05M to 5M, and the optimum salt concentration for growth is 2M to 3M. Because D. salina can grow in high salinity environments, where the other organisms survive hardly, large-scale culture of D. salina do not need expensively fermentive or other special equipments, suggesting that D. salina is a favorable host for producing pharmaceutical proteins.Though the technique of nuclejc transformation in plants has been developed and used widely, some problems in genetic information have not been resolved. For example, because the nucleic genome is so big andcomplicated that the integration sites and copies of foreign gene can not be controlled accurately, the expression of transferred genes is inefficient as a result of gene silencing or position effect. In nucleic transformation, furthermore, the transfer of multigene is difficult, and only after the prokaryotic genes undergo modification are they expressed in high plants.The chloroplast transformation may resolved these problems mentioned above. Compared with nucleic genome, the chloroplast genome is very small and easily to be manipulated genetically, and the foreign gene is site-direct integrated into the chloroplast genome through homologous recombination. This is helpful to locating the foreign gene introduced at a certain site where it can be expressed efficiently. Because there are many copies of chloroplast DNA and the chloroplast has a strong tolerance to accumulation of the products expressed by the introduced foreign gene, a high level of expression is often happened in chloroplast transformation. In addition, because of prokaryotic property of the chloroplast, the prokaryotic gene can be expressed in chloroplast without any modification and multigene can be simultaneously transferred in "polycistron", which is impossible in nucleic transformation.In order to form a chloroplast transformation system of D. salina, we have conducted some studies including its sensitivity to antibiotics, the activity of promoter, cloning of the chloroplast genes and construction of transformation vectors, so far a pilot transformation system of the D. salina chloroplast has been completed.Methods:The sensitivity of D. salina to seven antibiotics or herbicide used commonly in gene engineering was studied and the biological activity of atpA promoter from C. reinhardtii chloroplast was tested by using enhanced green fluorescent protein (EGFP) as a reporter. Primers were designed inthe conservative encoding regions according to the chloroplast genomes from four algae which have close genetic relationship with D. salina, and the sequences of 16S rRNA, chlL and chlN of D. salina chloroplast were cloned and sequenced, respectively. Three chloroplast transformation vectors including pDS16S-CAT, pTN1269-bar and pSP72-N5-bar-N3 were constructed, using!6S rRNA or chlN gene sequence as a homologous segment and CAT or bar as a selective marker gene, respectively. Foreign genes were introduced to the cells of D. salina by microprojectile bombardment method and a pilot chloroplast tran...
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