Optimization Of Green Algae Genetic Transformation And Fluorescence Analysis In Transformed DR5::GFP Chlorella Vulgaris

Single-celled algae is an ideal material for plant molecular biology researches. Shock transformation is the favour method for the researchers because of the simple operation and low cost. However, the strong electric field and long electric shock time in traditional algae shock transformation method would increase cell death and reduce transformation efficiency. In order to improve the efficiency of transformation, the algae shock transformation was developed. DR5 is a specific promoter which is induced by auxin and show high biological activity. DR5::GFP could be used to reveal the level of auxin in plant cells and tissues through laser confocal microscopy. In this study DR5::GFP was transferred into unicellular chlorella and the fluorescence was detected through laser confocal microscopy. To use transferred DR5::GFP green algae as the probe cells provide a good foundation for determination of IAA at single cell level.The main results were as follows:(1) A green algae cell transformation system was set up and the conditions of electric shock was developed. The better hygromycin concentrations were 25 mg/L and 100 mg/L when Chlorella vulgaris and Chlamydomonas reinhardtii transformants were screened respectively, and the better kanamycin concentration was 100 mg/L for chlorella transformants screening. The optimal electric field intensity was 0.8 KV/cm and 0.6 KV/cm in Chlorella and Chlamydomonas respectively. The optimal pulse time was 10 ms for both species. Making protoplasts and treating with 2-DG were able to significantly increase the transformation rates of green algae.(2) DR5::GFP was transferred into chlorella. The transgened DR5::GFP Chlorella was harvested through the optional electric shock after preparation of protoplasts 2-DG treatment.(3) IAA treatment was able to induce GFP expression. Fluorescence was detected through laser scanning confocal microscope after in vitro treatment with 4-5 μg/mL of IAA in transgened DR5::GFP Chlorella.(4)The cryopreservation conditions of transgened DR5::GFP chlorella was developed. The optimal cryopreservation temperature was -70℃ and the best cryoprotectant was 10% DMSO plus 10% sucrose after freezing at different temperature. Finally, about 34.68% of transgened DR5::GFP chlorella was survived after 7 days culture.