In order to prove that we have successfully constructed the CA-PRK, CA2-PRK overexpression system, and functioned in Phaeodactylum tricornutum. We conducted the following experimental verification: PCR to detect whether the recombinant plasmid contains CA-PRK and CA2-PRK genes; PCR to detect whether P. tricornutum contains our overexpression system after electroporation transformation; CLSM to observe the intracellular lipids and chlorophyll content of P. tricornutum. The results showed that the recombinant plasmid contained CA-PRK and CA2-PRK genes, and was successfully transferred into P. tricornutum. And the intracellular lipids content of the successfully transformed P. tricornutum showed significant changes.
Before electroporation transformation, we use the recombinant plasmid as a template for PCR detection to verify whether our recombinant plasmid contains the target gene. The results are as follows, the recombinant plasmid contains the target gene.
Fig 1. DNA validation
After electroporation transformation, we expanded the culture of Phaeodactylum tricornutum, and then extracted the DNA of Phaeodactylum tricornutum using the CTAB method. The DNA was used as a template to detect whether the gene of the recombinant plasmid was introduced into the chassis organism by PCR.
Fig 2. DNA validation
Protein was also extracted for WB validation.
Fig 3. Coomassie bright blue stain
Fig 4. Western Blot verify
Fig 5. mRNA level expression of target genes
The results showed that the Pha::CA-PRK-eGFP overexpression system and Pha::CA2-PRK-eGFP overexpression system we constructed were successfully tranferred into Phaeodactylum tricornutum.
We used seawater f/2 medium to cultivate transgenic algae strains, and tested many physiological and biochemical indicators. The test results are as follows.
In terms of growth, the growth of the algae strains transferred into the overexpression vector system was not affected, and there was no significant difference between their cell density and that of wild type algae strains.
Fig 6. Cell density
Chlorophyll fluorescence parameters
Fig 7. Photosynthetic rate on day 5 of incubation
Content of three metabolites
Fig 8. Lipids content
Fig 9. Glucose content standard curve and Carbonhydrate content
Fig 10. BSA standard curve and protein content
After testing, both transgenic algae strains were found to have significantly higher oil content. Pha::CA-PRK-eGFP has 2.3 times the oil content of the wild type, and correspondingly its protein content and polysaccharide content were reduced. The increase and decrease in the content of the three metabolites is in line with the widely accepted pattern.
Fig 11. The content of the three polyunsaturated fatty acids
The content of polyunsaturated fatty acids was found to be significantly higher by GC-MS analysis. The content of ARA and EPA were twice as high as that of wild type. DHA content also has some improvement. This makes sense for cost reduction of high value products.
LSM
Fig 12. LCM observation
An increase in the number of oleosomes in the transgenic algae strain can also be seen under LCM observationAn increase in the number of oleosomes in the transgenic algae strain can also be seen under LCM observation
Fig 13. Nitrogen and phosphorus content
From the nitrogen and phosphorus content assay, it can be seen that the rate of nitrogen and phosphorus uptake of the transgenic algae strain（Pha::CA-PRK-eGFP） was enhanced
The results showed that the expression levels of genes related to phosphorus transport and lipid metabolism were significantly up-regulated in one of the transgenic algae strains (Pha::CA-PRK-eGFP).
Fig 14. qPCR analysis of key genes
Based on the above indexes, we screened out a better algal strain (Pha::CA-PRK-EGFP) for waste water culture to verify its viability in wastewater and nitrogen and phosphorus removal capacity.
After testing, we found that the growth of transgenic algae was not affected under the waste water culture environment, and the accumulation of lipid was significantly increased.
Fig 15. Cell density
Fig 16. Nile red fluorescence content
Based on the results of gene level and protein level assays of Phaeodactylum tricornutum, we judged that the CA-PRK overexpression system and CA2-PRK overexpression system had been successfully introduced into Phaeodactylum tricornutum . The LSM observation showed that the overexpression system was successfully expressed in the sump organisms, and one of the transgenic strains showed a significant increase in lipid accumulation, which was twice that of the wild-type strain.