Application Of Ku Gene And Conditional Lethal Gene CodA In Gene Editing Of Chlamydomonas Reinhardtii

Chlamydomonas reinhardtii,as one of model species,is widely used in microalgae research.It is the only eukaryotic algae can be transformed in the three genetic systems of nucleus,chloroplast and mitochondria.Although the nuclear transformation system of Chlamydomonas reinhardtii has matured and exogenous DNA molecules can be efficiently integrated into the nuclear genome,this integration is mainly random insertion.Up to now,the targeted knockout and replacement technology of the nuclear gene of Chlamydomonas reinhardtii has not yet been established effectively.How to achieve the insertion,knockout and replacement of DNA in a specific region of the genome is the bottleneck for the gene edit of Chlamydomonas reinhardtii.Aiming at the low efficiency of homologous recombination against Chlamydomonas reinhardtii,we studied from two aspects:the Ku gene and conditional lethal gene codA,explored the effects of the endogenous Ku gene knockout/inactivation on homologous recombination of its nuclear genome and the possibility of using codA to reverse screening.The results showed that we could integrate the exogenous gene at the maa7 locus of the inactivated Ku gene algae,and initially completed the gene editing.The growth of codA transgenic engineering algae was basically unaffected,only died under the induction of5-FC,indicating the feasibility of reverse screening of Chlamydomonas reinhardtii.All of these laid the foundation for the establishment of the directional editing technology system of Chlamydomonas reinhardtii.The main work and results are as follows:1.According to the codon bias preference of Chlamydomonas reinhardtii to modify Escherichia coli codA gene,an optimized gene(crcodA)with a length of 1311 bp was obtained,and a pJ1D-crcodA expression vector containing a heat-inducible promoter was constructed and transferred into the nucleus of Chlamydomonas sp.CC849 by bead milling.The engineering algae codA1 and codA3 were obtained by PCR and sequencing.The results of real-time quantitative PCR showed that the expression level of codA gene in codA3 was higher than that in codA1,and the expression of codA gene in engineering algae was increased by about 1 fold after the heat induction.Using 5-FC as a lethal inducer was found that when 5-FC concentration reached 5 mg/mL,it would be toxic to the engineered algae,and the toxicity was related to the concentration of algal cells,only when the concentration of algal cells was less than 8×10~2 cell/m L,5-FC would cause the death of the engineered algae codA3 cells.The detection of chlorophyll content revealed that the engineered algae was significantly decreased after lethal induction of 5-FC,which was about 1 fold lower than that of wild type,but the heat induction did not significantly change the chlorophyll content of the engineered algae,indicating that heat-induced has little effect on lethal induction of 5-FC.2.To improve the lethality of codA in Chlamydomonas,the heat-inducible promoter Hsp70-RBCS2 was replaced by constitutive promoter PsaD,and the fusion expression vector of crcodA+crupp gene was further constructed.Firstly,according to the codon bias preference of Chlamydomonas reinhardtii to modify Escherichia coli upp gene,an optimized gene(crupp)with a length of 648 bp was obtained,and a pDb-crcodAupp expression vector containing a constitutive promoter was constructed and transferred into the nucleus of Chlamydomonas sp.CC849 by bead milling.The engineering alge upp1 and upp2 were obtained with the high-fusion expression of crcodAupp gene.The study of 5-FC lethal induction showed that when 5-FC concentration of 5 mg/mL would be toxic to the engineering algae,and the growth of engineered algae was significantly inhibited when the algae cell concentration was less than 1×10~5 cells/mL,and all engineered algae died when the concentration of algal cells was less than 1×10~3 cells/mL.The results indicated that the improved codA gene would cause completely death of Chlamydomonas reinhardtii under certein conditions(adding 5-FC),demonstrating that using codA for reverse screening in Chlamydomonas reinhardtii was feasible3.In the genome of Chlamydomonas reinhardtii,two Ku-like protein genes crKu70,cr Ku80 were predicted,and the possible crKu70,crKu80 inactivated mutant algae dKU70and dKU80 were screened from the Chlamydomonas mutant library.After staggering purification of possible inactivated mutant algae,their genotypes were detected by PCR sequencing.The results showed that reversed insertion of the exogenous resistance gene(CIB1 cassette)occurred in the eighth intron of the crKu70 genome in the dKU70 algae strain.And the resistance gene lost 265 bases at the 3?end when inserted;the positive insertion of the entire resistance gene occurred in the fifth exon of crKu80 genome in the dKU80 algae strain.The results confirmed from the gene level that the algae strains dKU70and dKU80 were indeed mutant algae with inactive crKu70,crKu80 inactivation.Due to Ku-like protein was related to DNA damage repair,algae strains dKU70 and dKU80 were sensitive to DNA damage agents,but dKU80 is more sensitive than dKU70.The results further validated that dKU70 and dKU80 are endogenous Ku-like gene inactive algae from functional perspective.Given the more obvious phenotype of dKU80,the algae was selected for subsequent gene editing studies.4.The homologous recombinant vector pMD19T-maa7LRhyg was constructed by selecting endogenous maa7 gene of Chlamydomonas reinhardtii as the target gene.The vector contained the recombinant unit consisting of a 1kb upstream of maa7,hygromycin resistance gene,and 1kb downstream of maa7.The recombinant vector was transformed into an algae strain dKU80,and transformants were obtained by screening with hygromycin and5-fluoroindole.PCR analysis of the transformants showed that the homologous recombinant unit successfully replaced at the maa7 gene in the dKU80 strain.The results preliminarily confirmed that endogenous Ku80 inactivated algae strain can perform directed gene editing at a specific location.In summary,on the one hand,in this study the engineering algae of the crcodA+crupp fusion expression gene were obtained by genetic engineering,which confirmed the feasibility of the codA gene as a reverse screening marker in the nuclear gene editing of Chlamydomonas reinhardtii.On the other hand,the inactivated mutant algae of endogenous Ku-like protein were obtained,which confirmed that the genome directed editing in crKu80inactivated algae could be initially achieved by way of homologous recombination vectors.This paper provided a beneficial exploration for the development of the homologous recombination technology of Chlamydomonas reinhardtii,and opened up a new way of thinking,which laid a foundation for the establishment of the directional editing technology system of Chlamydomonas reinhardtii.