Isolation Of Intact Chloroplasts From Dunaliella Salina And Construction Of Vector For Chloroplast Transformation

Recently, the research focusing on genetic transformation of algae has been carried out intensively, and some of them have been developed as new type bioreactors for the production of pharmaceutical proteins or vaccines. Compared to traditional transformational systems including microbial fermentation, transgenic animals and plants, transgenic algae have some particular advantages, for example, they are autotrophy and do not require expensive media or strict culture conditions. Because of these unique characteristics, the algae are developed to low-cost and safe bioreactors.Nowadays, studies for the genetic transformation of unicellular eukaryotic algae mainly focus on two approaches: nucleus and chloroplast transformation. Though the nucleic transformation in plants has been developed and used widely, some intrinsic problems in genetic information have not been solved. The nucleic genome is so big and complicated that the integration sites and copies of foreign genes can not be controlled accurately, leading to the inefficient expression of the foreign genes because of gene silencing.The chloroplast transformation may solve some of the problems mentioned above. Compared to nucleic genome, the chloroplast genome is smaller and easier to be operated genetically, and the foreign gene is site-direct integrated into the chloroplast genome through homologous recombination. Besides, there are many copies of chloroplast DNA(cpDNA) in single chloroplast, and the chloroplast has a strong tolerance to accumulation of the products expressed by the introduced gene, which will ensure the high efficient expression of the foreign gene. Meanwhile, the prokaryotic genes are efficient expressed in the chloroplast without any modification because of prokaryotic property of the chloroplast transformation, but not in nucleic transformation.Dunaliella salina (D. salina) belongs to Chlorophyta, Chlorophycease, Volvocales. Its shape and structure are very similar to Chlamydomonas reinhardtii (C. reinhardtii) except for lacking of cell wall. It has a large cup-shaped chloroplast about 50% of the cell volume. The cells are able to grow in extreme environments such as in a variety of sodium chloride concentration ranging from 0.05M to 5M, where other organisms hardly survive, so large-scale cultures of D. salina do not need expensive equipments, suggesting that D.salina is a favorable host for producing pharmaceutical proteins.In this study, after a chloroplast genome walking library was constructed, target homologous DNA fragments were obtained by using nested PCR, and finally a vector for chloroplast transformation was successfully constructed.Methods:1. Isolation of the intact chloroplast from D.salina and extraction of cpDNAThe cells grown at -27℃in UTEX medium containing 1M NaCl, and a light/dark regime of 12/12h was selected. Before harvesting, cultures were examined microscopically for contamination. Subsequently, exponentially growing cultures, about 7 days after inoculation, were harvested and broken through cell bomb. The intact chloroplasts were obtained by differential centrifugation and sucrose density gradient centrifugation. A modified scheme of SDS-proteinase K-phenol/chloroform/isoamyl alcohol was employed to extract cpDNA.2. Amplification of uncharacterized flanking regions of the chlN genechlN together with chlL and chlB are responsible for the biosynthesis of light-independent protochlorophyllide reductase (LIPOR) of the chloroplasts in D. salina cells. D. salina cells can also perform chlorophyll formation through light-dependent protochlorophyllide reductase (LDPOR) even without chlN gene. So the loci of the chlN genes were selected as the homologous recombination regions.2.1 Construction of chloroplast genome walking librariesFour chloroplast genome walking libraries DL, EL, PL and SL of D. salina were constructed after the cpDNA digested by Dra I, EcoR V, Pvu II and Stu I was ligated to designed adaptors.2.2 Amplication of uncharacterized flanking regions of the chlN genesPrimers used in the amplification were designed according to the upstream and downstream sequences of the chlN gene, and the chloroplast genome walking libraries were served as the templates. The nested PCR was selected for amplification of uncharacterized flanking regions of the chlN genes.3. Construction of vector for the chloroplast transformation and identification for result of transformationA new vector named as pMDko-bar for chloroplast transformation had been constructed after the homologous recombination fragments were obtained, and then the pMDko-bar was introduced into wild type cells of D. salina by electroporation.Result:1. Isolation of the intact chloroplast from D.salina and extraction of cpDNAResult of sucrose density gradient centrifugation revealed that a mass of chloroplasts were isolated. The integrity of the chloroplasts was confirmed with phase contrast microscope and electronic microscope. The results of examination for the concentration and purity of the cpDNA through an ultraviolet spectrophotometer and 1 % agarose gels were satisfactory.2. Amplification of uncharacterized flanking regions of the chlN geneThe result of nested PCR indicated that 600 bp and 3000 bp of fragments were amplified from the upstream chlN, and 650 bp of fragment was amplified from the downstream chlN through genome walking technique. The sequencing results showed that the amplified fragments were correct.3.Construction of vector for the chloroplast transformation and identification for result of transformationThe identification for vector pMDko-bar indicated that both the size and direction of the inserted fragment were correct. One week later, the transformed cells were able to grow under the select pressure of 0.2μg/ml PPT, while the negative control hardly survived. The above observation suggests that the target gene has been introduced into the chloroplast of D. salina cells.Conclusion:1. cpDNA with high purity has been extracted after intact chloroplasts wereisolated from D. salina cells.2. A vector pMDko-bar for chloroplast transformation has been successfully constructed after homologous fragments were obtained.3.identification results reveal that the target gene has been introduced into the chloroplasts of D. salina cells.