Transformation And Expression Of Exogenous SAD Gene In The Chlorella Pyrenoidosa And Its Effect On The Of Fatty Acids Composition Of The Genetically Modified Microalga

In order to explore the role of microalgae stearyl carrier protein desaturase(SAD)gene in the regulation of unsaturated fatty acid synthesis of Chlorella pyrenoidosa,and to enhance the synthesis of unsaturated fatty acids in algal cells,a foreign SAD gene,from Chlorella zofingiensis,was transferred into the chloroplast of C.pyrenoidosa,and the transformant was successfully obtained through antibiotic resistance screening and homogenization.PCR and fluorescence quantitative PCR analysis of the transformant were carried out to confirn the gene transformation.The oil content and fatty acid composition of the transformant were analyzed.Chloroplast gene transformation was successfully applied first time by gene gun method in the microalga C.pyrenoidosa in the paper,and the results are benefit in understanding the mechanism of fatty acid synthesis of microalgae.The main results of this paper are as follows:(1)The existence of the chl L gene sequence was verified in the chloroplast genome of C.pyrenoidosa,which would be used as homologous DNA fragment.The antibiotic sensitivities of C.pyrenoidosa were evaluated among six antibiotics,and the results showed that Chlorella pyrenoidosa was completely killed in about eight days when the concentration of streptomycin was 200?g/m L,so aad A gene was selected as a marker gene.(2)The chl L gene sequence was successfully constructed in the plasmid as the homologous fragment,and the promoter atp A and terminator rbc L of Chlamydomonas reinhardtii chloroplast were used for the expression of SAD gene.(3)The SAD gene was transferred into the chloroplast of C.pyrenoidosa by gene gun bombardment method,and the transformant was selected.(4)PCR and fluorescence quantitative PCR were used to detect wild C.pyreonosa and transformant.The results showed that the foreign gene wassuccessfully integrated into the chloroplast genome of C.pyrenoidosa,and the quantitative expression of SAD gene showed that the relative expression level of SAD gene was 1.65 times(p <0.01).(5)The content of unsaturated fatty acid increased from 71.57% of wild algae to78.66% of transformed algae,especially C17: 1,which indicated that the SAD gene from Chlorella pyreonosa played a role,and made the unsaturated fatty acid content increased significantly.(5)The content of unsaturated fatty acid increased from 71.57% of wild algae to78.66% of transformed algae,especially C17: 1,which indicated that the SAD gene from Chlorella pyreonosa played a role,and made the unsaturated fatty acid content increased significantly.