Preliminary Study On Transformation System Of Genetic Green Algae

In this paper, the transformation system has been preminary studied in Ulva lniza and Chlorella protothecoides: 1 ) Screening oil production and molecular identification of microalgae; 2), Determine the selection marker of Ulva lniza and Chlorella protothecoide 3) Preliminary study on transformation system of Ulva lniza and Chlorella protothecoide; 4) Cloning and expression PEPC gene of Chlorella protothecoid in E.coli. All theses works laid a foundation for the building of genetic transformation model in Ulva lniza and Chlorella protothecoide.Firstly, the study of the microalgae oil fluorescence detection methods and molecular identification of microalgae were discovered. Using Nile Red, BODIPY which were stained specificity of oil to establish the rapid detection of microalgae, which can be used as indicators of the lipid lever of genetic microalgae. In this way, we selected one kind Chlorella sp. Which contended 21.66±0.49% lipid. After that, Chlorella sp.was identified to be Chlorella protothecoide.with the rbcL and ITS genetic markers.Secondly, the antibiotics and herbicides on Ulva sp. and Chlorella protothecoide was tested as selection marker. Herbicide (Basta) had strong lethal effect on Ulva linza,Ulva clathrata, Ulva prolifera 12.5μg/ml herbicide (Basta) could be proper selectable marker in protoplast transformation system. Different strains of algae have different levels of herbicide tolerance to Basta at different growth stages .With the growth of algae, algae had increasing tolerance to Basta, and showed species differences.The order of sensitivity to Basta was as follows: Ulva linza,Ulva prolifera, Ulva clathrata. By studying sensitivity of Chlorella protothecoide to six kinds of antibiotics and herbicides (Basta), Basta could be better select marker considering vector and economic costs, we chosed bar as the resistance selection marker. Five days after culture, 20μg/ml of Basta can be a good selection marker of Chlorella protothecoide.Thirdly, genetic transformation system of green algae was further studied. Chlorella sp. has been established succesefully genetic transformation system with PEG, electroporation, gene gun method, which has great significance to our study. Meanwhile, based on the original study in the laboratory, the PEG transformation of Ulva sp. method was optimized with the PH, PEG₆₀₀₀ concentration, plasmid content, the average relative conversion rate could be reach 38.58%. The transferred seedlings continuously were cultured in VSE with Basta. Besides, and the transferred seedlings resisting to Basta were pick up for PCR molecular identification with bar gene sequence. The result showed the DNA information from the transferred seedlings cultured both for 1 month and 12 months was positive,which was consisitent with the sequences in GenBank . The optimal conditions in a final concentration of 20% PEG₆₀₀₀, PH8.0 , 20μg plasmid DNA into 2×105 cells can achieve better results, the average conversion rate could reach at 38.58%.Finally, the PEPC of Chlorella protothecoides was cloned.And bioinformatics analysis was done on the level of the nucleotide and amino acid sequences .In addition, further construction of pGEX-4T-1-PEPC was expressed well in E. coli. Experimental results showed that PEPC from Chlorella protothecoides successfully expressed in E.coli. The protein content increased to 130% and the total lipid was reduced to 45.1%, while PEPC activity in transformed strains increased 127% than the wild strain.These studies showed the increase in PEPC activity controlled the direction of flow of carbon. The protein/lipid ratio verified from 3.3 to 9.8,which protein synthesis was increased and the lipid synthesis was inhibited. PEPC was a key enzyme for the regulation of protein and lipid content.All these works builded a foundation for the establishment of genetic engineering of microalgae.