Transcriptional Activity Analysis Of Chlorovirus Promoters In Chlamydomonas Reinhardtii

The chlorella virus-derived promoter has the characteristics of initiating gene transcription in both prokaryotes and eukaryotes.This experiment uses a promoter sequence from chlorella virus and has an emphasis on control activity in E.coli to construct Chlamydomonas reinhardtii Transformation vector.The ble-gfp selection marker-reporter gene fusion gene formed by fusion of the bleomycin resistance gene sh ble and the green fluorescent protein gene gfp was placed under the control of the chlorella virus promoter,and a promoter-free and endogenous rbcS2 Promoter-controlled positive and negative control transformation vectors.The above transformation vector was introduced into the cell wall-deficient Chlamydomonas reinhardtii strain cc503 by electric shock,and the plate and liquid were screened with 7.5 ?g/mL and 4 ?g/mL bleomycin to obtain a series of sh ble resistant algae;The negative control transformation vector did not yield any resistant algae.After PCR amplification,it was found that the ble-gfp fusion genes under the control of N63,N80,N99,N35 promoters derived from chlorella virus were inserted into the sh ble resistant algae cell genome.The qRT-PCR method was used to detect the abundance of sh ble transcription text controlled by the viral promoter and the rbcS2 promoter,and the GFP expression was observed by laser confocal microscopy.With normal starting activity.A recombinant plasmid containing the gpd1 gene was constructed from the promoter with the strong promoter activity selected above,and also transformed into Chlamydomonas reinhardtii cells by electric shock transformation method.The gene inserted into Chlamydomonas reinhardtii was detected by PCR technology and stained with Nile Red.The detection of Chlamydomonas reinhardtii strains containing the gpd1 gene method showed that the oil content of Chlamydomonas reinhardtii strains did indeed increase.The specific experimental results are shown below:1.Construction of pBR11-N35,pBR11-N63,pBR11-N80,pBR11-N83,pBR11-N96 and pBR11-N99 plasmids,constructing positive and negative control plasmids pGO24,pGO25.These constructed plasmids were transformed into Chlamydomonas reinhardtii cells byelectroporation,and positive algae strains were screened with 7.5 ?g/mL of bleomycin resistance.As a result,pBR11-N35,pBR11-N63,pBR11-N80,and pBR11-N99 were found can grow on bleomycin-resistant plates.2.Through PCR detection,we found that sh ble resistance selection marker genes under the control of chlorella virus-derived promoters N35,N63,N80,and N99,and the GFP reporter gene were inserted into the genome of Chlamydomonas reinhardtii,indicating that the algal virus promoter,like the rbcS2 promoter,has normal promoter activity in Chlamydomonas reinhardtii.The qRT-PCR method was used to detect the abundance of the sh ble transcription text controlled by the viral promoter and the rbcS2 promoter.It was found that the expression of N63 and N35 promoters was up-regulated relative to rbcS2,and the activity of N63 was the strongest,reaching 1.78 times of that of rbcS2 promoter.It indicated that the Chlorella virus promoter and the rbcS2 promoter had normal priming activity in Chlamydomonas reinhardtii.3.Using the N63 promoter to construct a plasmid containing the gpd1 gene,transform it into Chlamydomonas reinhardtii cells by electric shock transformation,and screen for positive algae strains with 7.5 ?g/mL bleomycin resistance,and find N63-gpd1 and rbcS2-gpd1 Transformants were able to grow in bleomycin-resistant medium.4.The genome of the transformed algae strain was extracted and tested by PCR.Both the N63-gpd1 and rbcS2-gpd1 transformants could amplify the target gene.5.The Nile Red staining method was optimized,and it was found that the Chlamydomonas reinhardtii cells had a final Nile Red-Acetone concentration of 0.3 mg/ml,and the oil droplets were clearer under the condition of room temperature staining for 20 minutes in the dark.6.The oil content of Chlamydomonas reinhardtii transformed strain was significantly higher than that of wild type strain CC503(p<0.05),and the content of transformed strain was1.23 times higher than that of wild type strain.