Construction Of Double Selective Transformation Vector PchlN-CAT-BAR And Transformation For Chloroplast Of Dunaliella Salina

Dunaliella salina(D. salina) is a unicellular eukaryotic green algae which is ellipse or pear-shaped with two equal length flagellum and can survive in medium with salinities ranging from 0.05 to 5 M sodium chloride. It is suitable to chloroplast transformation because it contains a large cup-shaped chloroplast covering about 50 % of the cell volume and lack of cell wall outside the cell membrane. At present in the fields of using algae as a transgenic hosts, the study on Chlamydomonas reinhardtii which is a model organism has tend to mature, while the same research on D. salina, another similar Chlamydomonae algae, has developed step by step.In the past studies, exogenous gene has been expressed in the nucleus of D. salina successfully. But we also found that the expression of a exogenous gene in the nucleus was often difficult, unstable and low, that restricting the practical application of nucleic expression system. Compared with nucleic system, chloroplast expression system has some certain advantages:because chloroplast genome is smaller and the genetic background is clearer, the foreign gene can be inserted into the genome by homologous recombination and site-specific integration in which the insertion site can be controlled accurately; because the genes of chloroplast are arranged in polycistronic form, multiple foreign genes can be expressed simultaneously in chloroplast system; because chloroplast has many copies of DNA, the expression of foreign gene can be more efficiently; and chlorplast system also has advantages such as maternal inheritance, low-risk for gene flow; though chloroplast is near to prokaryotic cell, the protein expressed in chloroplast can be modified by other processing except glycosylation. Thus, chloroplast transformation is an important field of D. salina transformation. In this paper, exogenous selective marker genes were transferred and expressed in the chloroplast of axenic purified D. salina by homologous recombination.To establish the transformation system of D. salina chloroplast, it is very important to construct a high efficiency chloroplast expression vector. The chlN gene, along with chlL and chlB gene, coding the light-independent protochlorophyllide reductase (DPOR), is mainly involved in the synthesis of chlorophyll in the dark. If anyone of these three genes is inactivated, the synthesis of chlorophyll under dark conditions will be blocked. But the blocking does not affect the normal living of D. salina, just because it can still synthesize chlorophyll in light though light-dependent protochlorophyllide reductase (LPOR). Therefore, the chlN gene is an ideal integration target of chloroplast homologous recombination. In this paper, we constructed a chloroplast transformation vector pchlN-CAT-BAR for D. salina, which used chlN gene and its up-and down-stream sequences for 4.0 kb as homologous segment, and the chloramphenicol acetyltransferase gene(cat) and the herbicide phosphinothricin (PPT) resistant gene(bar) as selective marks, and aptA promoter and rbcL terminator as regulatory elements. The vector was transferred into the cells of normal D. salina by microprojectile delivery under 100 psi helium pressure and then a homologous replacement occurred between plasmid vector and chloroplast genome, resulting in a site-specific integration through the insertion of the exogenous cat and bar genes into the chlN gene of the D. salina chloroplast genome. Before transformation, centrifugation, dilution and antibiotics were used to prepare axenic D. salina cells and the working concentration of antibiotics were determined by the death of bacteria and survival of D. salina. The transformants were selected firstly by chloramphenicol and secondly by PPT, and the cat gene was used as a major selective mark while the bar gene as an assisted selective gene. The Cell Disruption Bomb was used to break the cells of stable transformants, and the intact chloroplast was isolated by sucrose density gradient centrifugation, and the chloroplast genomic DNA was extracted by SDS alkaline lysis method. The integration of cat gene and bar gene chloroplast genomic DNA of the transformants, was identified by PCR amplification and sequencing.The results showed that the transformants of D. salina survived under the selection pressure of chloramphenicol at 200μg/ml, while the wild type cells died about 12 days. Besides, the stable transformants were obtained by the selection pressure of 4μg/ml PPT for 3-5 generations, suggesting that the transformants have resistances to both chloramphenicol and PPT. From this, we can get it is available to construct transformation vector for D. salina chloroplast using the chlN gene as homologous segment, and both the cat and bar genes as selective marks. Moreover, it can also provide a precondition of using chlN gene as homologous fragment to express foreign protein in the chloroplast of D. salina.