Study On Mechanism Of Mutagenesis Induced By Heavy Ion Beam Irradiation And Mitochondrion-related Function Based On Saccharomyces Cerevisiae Model

Heavy ion beam irradiation-based mutation breeding has the advantages of high mutation rate,wide mutagenesis spectrum,high genetic stability of mutants,easy generation of new mutations,and the tendency to expand genetic diversity.It has broad application prospects in microbial breeding.However,the blindness of mutation breeding technology are also shortcomings for heavy ion beam irradiation-based mutation breeding.In the future,modules such as optimization of radiation parameters,prediction of mutagenesis populations,high-throughput screening of mutants,effective identification and verification of positive mutation sites in mutant,and targeted integration of positive mutation sites will be integrated into heavy ion beam irradiation-based mutation breeding workstation.This will bring into full play the strengths and effectively make up for the weaknesses,and further promote the efficient and high-quality operation of heavy ion beam irradiation-based mutation breeding practices.The realization of above vision involves a series of basic research needs.In this work,we selected Saccharomyces cerevisiae as the cell model and focused on three progressive themes important for achieving efficient mutagenesis:dose-survival effects,molecular mutation spectrum,associations between phenotype and genotype.The following research was carried out.The determination of dose-survival effects is a prerequisite step for the study on heavy ion beam irradiation mutation breeding.We selected X-rays as the source of ionizing radiation and S.cerevisiae as a single-cell microbial model,and improved the OD₆₀₀-based method and established a 96-well method to determine the survival fraction of single-cell microbe induced by ionizing radiation.For the OD₆₀₀-based method,cells were exposed to various X-ray doses and inoculated into fresh medium:a lower biomass accumulated,indicating fewer surviving cells within the investigated dose range（0–100 Gy）.For the 96-well method,diluent containing～0–100 cells were equally divided into 96 droplets and respectively inoculated into 96 wells containing200μL of broth:fewer wells without S.cerevisiae clones indicated more surviving cells after 48 h of incubation.Corresponding quantitative systems were established for two methods according to exponential function and Poisson distribution probability density function,respectively.Both of them were sensitive and reliable.The OD₆₀₀-based method is simple and fast,and the 96-well method simplifies the counting process.In addition,comparisons of OD₆₀₀-based,96-well,and plate-counting methods indicated a higher rate of repair for S.cerevisiae in liquid culture than that on agar.In addition,different batches of the experiments have shown that after heavy ion beam irradiation treatment,the timeliness of the plate count may have a greater impact on the measured value of the survival fraction.We aimed to verify the saddle-shaped dose-survival effect of microbes in response to heavy ion beam irradiation,and further to determine the radiation parameter that affects saddle shape formation,and the relationship between the saddle region and the positive mutation rate.A bibliometric analysis was performed based on literature containing the dose-survival effect of microbes in response to heavy ion beam irradiation.The data of particle energy,ionic types,irradiated microbe species,survival curves,and maximum positive mutation rates were assembled.The predominant articles,authors,and institutions that reported the dose-survival effect of microbes in response to heavy ion beam irradiation proposed the saddle-shaped survival curve.It was customarily low-energy heavy ion beam irradiation that induced the saddle-shaped dose-survival effect.However,few studies also reported the saddle-shaped dose-survival effect of microbes induced by moderate-energy heavy ion beam radiation.Thus,the formation of saddle-shaped dose-survival effects may involve the comprehensive effects of more complex factors.In addition,the saddle-shaped dose-survival effect was general among～30-genera microbes.More importantly,peaks in most of the saddle curves contained the survival fractions within 10–30%.About 87%of the maximum positive mutation rates were associated with the peak in saddle curve,and 58%were located near the peak point.Consequently,saddle region is always accompanied with high positive mutation rates.To reveal the microbial molecular mutation spectrum induced by heavy ion beam irradiation,mitochondrial dysfunction in S.cerevisiae was selected as a marker of ion penetration following carbon ion beam irradiation.Respiration-deficient mutants were screened.Following confirmation of negligible spontaneous mutation,eight genetically stable S.cerevisiae respiration-deficient mutant strains and a control strain were re-sequenced with>200-fold read depth.Strategies were established to identify and validate the particular mutations induced by carbon ion beam irradiation.In the nuclear genome,carbon ion beam irradiation mainly caused base substitutions and some small（<100 bp）insertions/deletions（InDels）,which were widely distributed across the chromosomes.Although mitochondrial dysfunction was selected as a screening marker,variants in the nuclear genome were detected at a high frequency(10^-7)relative to spontaneous mutations(10^-9).The transition to transversion ratio for base substitutions was 0.746.The relative number of single base substitutions and small insertions is about15:1.Small InDels are mainly<4 bp.In the mitochondrial genome,there were very large deletions including substantial gene areas,resulting in extremely low read coverage.Meanwhile,every mutant possessed a distinctive mutation pattern,for both the nuclear and the mitochondrial genomes.Damage repair characteristics induced by heavy ion beam radiation will reflect the biological basis of its mutagenic characteristics to a certain extent.We used transcriptomics to analyze the damage repair response in S.cerevisiae following semi-lethal heavy ion beam irradiation and X-ray irradiation,which both induced a significant number of DNA double-strand breaks（DSB）.Our analysis of differentially expressed genes（DEG）at consecutive time points post-irradiation revealed that upregulated genes were significantly enriched for gene ontology（GO）and Kyoto encyclopaedia of genes and genomes（KEGG）terms related to damage repair response.Based on the number of DEGs,their annotation,and their relative expression,we established that 75 min post-irradiation was the key time point of the damage repair response at the transcriptional level.A comparative transcriptomics analysis on the damage repair response induced by heavy ion beam and X-ray radiations was performed at 75 min post irradiation.Although these two radiations have different properties,we found a significant overlap（>50%）for the DEG associated with five typical DNA repair pathways.In both cases,homologous recombination repair（HRR）was identified as the predominant repair pathway.Nevertheless,when we compared the relative enrichment of the five DNA repair pathways at the key time point of the repair process,we found that the relative enrichment of HRR was higher after heavy ion beam irradiation than after X-ray irradiation.Additionally,the peak stage of HRR following heavy ion beam irradiation was ahead of that following X-ray irradiation.HRR is the main pathway of DNA DSB repair in S.cerevisiae.We considered that the fact that heavy ion irradiation corresponds to an earlier HRR pathway response is a compensatory response of the cell to more DNA DSBs.Although multi-omics studies at the population level are favored based on the dimensionality of information acquisition,it has been barely reported in studies on mutation.In this work,combined genome,transcriptome,and metabolome analysis was performed for eight S.cerevisiae mitochondrial respiration-deficient mutants.Each mutant exhibited a unique nuclear genome mutation pattern.Nuclear genome mutations,and thus potentially affected genes and metabolic pathways,showed a co-occurrence frequency of≤3 among the eight mutants.For example,only a lipid metabolism-related pathway was presumably affected by the nuclear genome mutations in one of the mutants.However,large deletions in the mitochondrial genome was common characteristic among the eight mutants.Meanwhile,nuclear genomes contained scanty mitochondrial respiration-related genes that were potentially affected by verified mutations.These results suggested that variants in the mitochondrial genome may be the main drivers of respiratory deficiency.At the transcriptomic level,lipid metabolism was the most significantly enriched KEGG pathway for DEGs co-occurring in both≥4 and≥5 mutants.Any identified DEG enriched in lipid metabolism showed the same up-/down-regulated pattern among nearly all eight mutants.Furthermore,the 126differentially expressed lipid species identified by lipidomics analysis also showed the same up-/down-regulated pattern among nearly all mutants investigated.It can be conservatively inferred that the similar change pattern of lipid metabolism in the entire investigated mutant population was attributed to mitochondrial dysfunction.The change spectrum of lipid species was presented,suggesting that the number and change degree of up-regulated lipid species were higher than those of down-regulated lipid species.Additionally,the main energy storage lipids increased in content and the important plasma-membrane phospholipid compositions varied in the relative proposition.In summary,we first conducted methodological and metrological studies on the microbial dose-survival effects induced by heavy ion beam irradiation.Then,at the S.cerevisiae mutant population level,a genome mutation spectrum induced by heavy ion beam irradiation（under particular radiation parameter in our study）was drawn,and its mutagenic characteristics were visually presented.The biological basis of heavy ion beam irradiation-induced mutation was revealed from the perspective of damage repair in S.cerevisiae.Finally,a multi-omics analysis was performed on the mutant population,and the association study of population-shared phenotype to genotype and population-shared genotype to phenotype was realized.We hope that these studies will further complement the research in mechanisms of heavy ion beam irradiation mutagenesis and provide guidance for efficient mutation breeding practices.