Strategies To Improve The Expression Of Exogenous Genes In Dunaliella Salina Cells

Objective:In order to improve the expression level of exogenous gene in the Dunuliella salina(D.salina),this paper try to establish a new transformation method for the D.salina transformation by the salt gradient.Based on this method,we made a deeply study of strategy feasibility of combination of membrane peptides mediated or nucleus localization peptide and recombinant plasmids to further improve the expression amount of exogenous gene in D.salina cells.In addition,the genetic stability of the transformant has been analyzed and finally to obtain the stable high expression of foreign genes of D.salina strains.Method:Using the characteristics of D.salina to adapt to different salt concentrations,when the high salt concentration(1.0M)transiently changes to a low salt concentration(0.1M),the foreign genes can be introduced into the D.salina through holes formed by the osmotic pressure difference.After the foreign material enters the cell,it is motivated with fluorescence of different wavelengths,and the presence and intensity of fluorescence at the nucleus of the cell are observed under a fluorescence microscope.The negative group without foreign substances was used as a control,and the same operation was performed in the same way.In order to obtain the best transformation efficiency,this paper has studied different conversion parameters,and optimized the four parameters that affect the conversion efficiency(conversion time,salt gradient,dye dose and Triton X-100 concentration).The influence of the conversion parameters on the conversion efficiency finally determines the optimal condition parameters of the highest conversion rate.Under this optimal condition,the recombinant vector(p CAMBIA1303),which containing a foreign gene,and the cell-penetrating peptide(TAT)or nuclear localization peptide(SVT40)have been transformed into D.salina cells.The plasmids is transformed with the nuclear localization peptide in order to obtain higher transformation efficiency and increase the expression level of the target gene.The experiment was provided with a negative control group(adding other plasmids for transformation)and a blank control group(without plasmids for transformation operation).After exogenous recombinant DNA was transformed into salt algae,cells were collected by centrifugation after 72 hours of culture.Histochemical staining and PCR amplification were used to analyze the expression of the foreign gene(GUS gene)in D.salina cells.After the cells were stained,they were observed and photographed under high magnification.The PCR products were identified by 1%agarose gel electrophoresis and sequencing.At the same time,the transformed cells were analyzed by RT-PCR to identify the expression of the foreign gene(GUS gene)at the transcription level.Transformed algae strains with the highest expression of foreign genes were selected from different groups(individual plasmids transformation group,plasmids combined TAT group,and plasmids combined SVT40 group)by fluorescent quantitative PCR.In addition,Western blot was used to analyze the protein product of the highest expressing strain to identify the expression level and biological activity.Genetic stability analysis was performed for the highest expressed strain for 3-6months,and genetically stable algal strains with high expression of foreign genes were screened.Results:Using the change of salt gradient,a salt gradient self-transformation method of salt algae was successfully established.When the high salt concentration is changed to the low salt concentration,the foreign material can be transferred into D.salina cell spontaneously.The results show that after the foreign material is introduced into the cell,the dye position in salt-algae cell is checked by different fluorescence.It showed strong red and green fluorescence under the fluorescence microscope.And also,the cells in the negative control group did not emit this fluorescence.Thereafter,the results showed that the exogenous substances were successfully introduced into D.salina cells.Through optimization studies of different transformation parameters(including transformation time,salt gradient,dye amount,and Tritonx-100concentration,etc.),the results show that among the four transformation parameters,the change in salt concentration has the greatest effect on the transformation results.With the decrease of salt concentration,the number of transformed cells increased significantly,but if the salt concentration is less than 0.1M,the transformation cannot be counted due to cell rupture and transformation failure.When the salt concentration is greater than 0.5M,the number of transformants appears to decrease significantly.When transformation time from 60s to 120s,the number of transformed cells increased significantly.But after the time exceeded 120s,due to the large number of cells ruptured,the number of transformed cells did not increase accordingly.The number of transformed cells also increases significantly as the 0.1%Triton X-100 concentration increases from 2?l(0.0005%concentration)to 10?l(0.001%concentration).When the amount of 0.1%Triton X-100 exceeds 0.001%,the number of transformed cells did not increase correspondingly due to the rupture of large number of cells.When fluorescent dye(EB,1.25 mg/ml)increased from 15?l to 30?l,the number of transformed cells increased significantly with the increase of concentration,but after30?l,the cells did not increase correspondingly due to the rupture of cells.After optimizing the above four parameters,the optimal transformation conditions were finally determined:When 1 ml of 0.1M saline solution medium(10~6-10~7/ml cell concentration),add 0.1%Triton X-100 10?l(0.001%concentration)and 30?l of1.25mg/ml EB,obtain the highest conversion efficiency at 120s transformation time(close to 100%).With this conversion condition,we carried out subsequent conversion work.After the exogenous plasmids were transformed into D.salina,the results showed that the positive cells could see blue or light blue color under the microscope,while the cells of the negative control group and the blank control group did not have any color reaction,which showed light yellow or yellowish brown.The results o f PCR analysis showed that the GUS gene fragment with a size of 1807bp was successfully amplified in different transformed algae strains,but no bands were displayed in the negative control group and the blank control group.The target gene was successfully transferred into D.salina cell.Histochemical staining results showed that the exogenous plasmid combined with the transmembrane peptide were also transformed into the D.salina cells.The highest expression algae strain was obtained by fluorescence quantitative PCR.Compared with the transformed plasmids group alone,the expression level was significantly increased at the transcription level,reaching 2.2to 2.8 times.However,Western blot results showed that no difference in protein expression was observed in the transformed lines,which may be related to the greater cell damage effect.After the plasmids combined with the nuclear localization peptide was transformed into the D.salina,the algal strain with the highest expression was obtained by fluorescent quantitative PCR screening.Compared with the transformed plasmid group alone,this algal strain was found not only to increase the transcription amount of GUS gene by 3.2 to 5.7 times at the transcription level,then due to the virus outbreak of novel coronavirus,the time of Western blot experiment was greatly limited,and there was no protein expression in the results,which was a pity of this study.In addition,the transgenic strains with the highest expression of exogenous genes also needed more than 3 months to analyze the stability at the transcription level and translation level,so as to obtain short-term stable genetic algal strains.However,it can be seen from the experiments that exogenous recombinant plasmids combined with nucleotide peptide can significantly improve the expression of exogenous genes,which lays a foundation for the future production of exogenous protein recombination.Conclusion:The self-transformation method has been successfully established for D.salina transformation,and the different transformation parameters have been optimized to obtain the best transformation efficiency.The new method has the advantages of simple operation,economical and practicality,no need for expensive instruments and experimental consumables,that can realize the efficient transformation of D.salina cells,and also provides a powerful tool for the genetic engineering research of the D.salina.The method of using an exogenous plasmids combined with a nuclear localization peptide can significantly increase the expression amount of the target gene in D.salina cell.The nuclear-localized peptide-mediated method is an attempt to successfully break through the current technical bottlenecks of the D.salina expression system,which provides a reference for recombinant production of foreign proteins using D.salina in the future.