Transformation Of NP-1 Gene In Chlorella Ellipsoidea Mutant And Sequencing Of ESTs In Dunaliella Salina

The defensins are kinds of antibiotic short peptides that produced by organisms against the invasion of pathogens. Because of the broad-spectrum antimicrobial activity and microbes are uneasy to produce resistance, defensins have an optimistic prospect of application in medicine. However, the low concentration of the neutrophile in organisms and the high price of extracting the peptides from organs make it necessary to produce defensins by genetic engineering method to enlarge their application field.Chlorella ellipsoidea is a genus of green eucaryotic algae and can be used as the receptor to express the peptide that post-translation modification is needed. In our project, an NP-1 gene expression vector which contains two selection marker genes NPTII and nitrate-reductase gene (NR gene) was constructed. The NP-1 gene was transformed into the nitrate-reductase deficient mutant of C.ellipsoidea nrm-4 via electroporation. The positive transgenic algae that can express the target peptide were obtained. Using NR gene as a selection marker makes the NR deficient mutant C.ellipsoidea a potential bioreactor for large-scale production of foreign proteins.Dunaliella salina is a kind of eucaryotic algae in mainland salt-water and coastal lakes. It is the most halotolerant algae in the world. D. salina has simple cell structure, short life cycle, easy handled and can be rapidly cultivated. The unique ability of D.salina to proliferate over the entire range of salinities (from 0.05 to 5M NaCl) makes it an ideal organism for the research of the resistance of cells on the stress conditions. Our project is the sequencing of the D.salina cDNA library on large scale. So far, about 4200 clones were sequenced and analyzed in our laboratory. The research contents and results in detail state as follows:1. A neutrophile peptide eucaryotic expression vector was constructed. The vector mainly consists of NPTII gene, NR gene and NP-1 gene. And what's more, the latter two genes are promoted by the same NR promoter cloned from C.ellipsoidea.2. The eucaryotic NP-1 expression vector was transformed into the NR deficient mutant of C.ellipsoidea nrm-4 via electroporation. The PCR analysis and the antagonistic...