Construction Of Functional Gene Mutant Strain Of Chlamydomonas Reinhardtii And Its Efficient Screening Technology

Chlamydomonas reinhardtii is a single-celled eukaryotic algae that has a short generation time,flagellation,sexual or asexual reproduction,and has been used as a model organism for cell and molecular biology research.Chlamydomonas reinhardtii can be used for photosynthesis.It is called "photosynthetic yeast".It is not only a model organism for studying photosynthesis mechanisms,but also a green cell factories for recombinant protein drug.Mutant librarie is the most important resource and platform for model species.However,there are still some problems for constructing the C.reinhardtii mutant library.Excessive null mutants and the difficulty of identification of insertion sites affect the efficiency of database construction.In order to solve these problems,the research proposes a method which efficiently obtain gene mutant by transforming the reconstructed drug resistance cassette and identify the insertion sites by using the 3' RACE.The specific results are as follows:(1)Obtaining the C.reinhardtii gene mutant: the ble gene resistance cassette lacking the 3'UTR was designed and constructed,and after transformation,the mutant was successfully obtained by screening plates with zeocin-resistant.(2)Obtaining the gene insertion information of the mutant: extracting the mutant RNA for RT-PCR and obtaining the ble gene and its ligated sequence.Confirming that the ligated sequence belongs to the C.reinhardtii genome sequence after sequencing alignment,and successfully predicting the location of the gene insertion.(3)Verification of the gene insertion position of the mutant: according to the predicted gene,primers were designed to amplify the genome of the mutant,and the obtained target fragment was significantly larger than the wild-type result.Sequencing further confirmed that the drug resistance expression cassette was inserted into the gene,which was consistent with the predicted result.(4)Small-scale experiments confirmed the feasibility of the method,and gene insertion information of 20 mutants was obtained: using the experimental method established above,gene insertion information of 20 mutants was obtained.Among them,the insertion position of the drug resistance cassette of 17 mutants was predicted to be within the annotated gene,and the effective mutant accounted for more than 85%.According to the prediction information,PCR was used to identify 10 mutants inserted into the gene coding frame by the resistance cassette,and 8 of them were confirmed to have the correct insertion position,with a positive rate of more than 80%.The accuracy of the prediction information was confirmed to be very high,and the method was stable and reliable.In summary,the research established an efficient method to obtain and identify functional gene mutants of C.reinhardtii,which solves the problems of large number of invalid mutants and difficult identification of insertion sites in the process of database construction and provides reference for the establishment of large-scale database of Chlamydomonas reinhardtii mutant library.