| Heavy ion beam irradiation can cause DNA single strand break,double strand break or DNA cluster damage in Saccharomyces cerevisiae and mutations such as DNA base conversion,transversion,base fragment insertion or deletion can be induced.When one or more of these mutations happen in mitochondria,damages will be occurred.Yeast cells will response like loss respiratory function,slower growth and reproduction,smaller colony morphology,and altered intracellular carbon cycle pathways.In this study,S.cerevisiae BY4743 in the logarithmic phase was exposed to carbon ion beam,and 10 stable mitochondrial damage mutants were screened by triphenyltetrazolium chloride(TTC)plate.By detecting the proliferation rate and respiratory intensity,five mutants with obvious mitochondria damage were secondary screened.The mitochondrial membrane potential were decreased in these mutants.Genomic sequencing confirmed that large fragments of mitochondrial DNA deletion is the import reason of changes in the phenotype of mutants.Through the combined analysis of lipid metabolomics and transcriptomics,it is preliminarily confirmed that the up-regulated expression of YRO374 W is the main cause of the increase in the relative content of mutant triglyceride(TG).The expression of YBR029 C significantly related with the content of phosphatidylinositol(PI)and phosphatidylserine(PS).In ResD-4,the down-regulated expression of YNL169 C directly led to a decrease in the relative content of phosphatidylethanolamine(PE).In ResD-1,the YJL134 W significantly up-regulated and YOL011 W significantly down-regulated were possibly leading to metabolic pathways change of sphingolipids and glycerophospholipids.The results preliminarily revealed the mechanism of the changes of cell lipid components induced by heavy ion beam irradiation in mitochondria dysfunction mutants.And considering the importance of S.cerevisiae as a model organism,the results of this study can also be references for higher eukaryotic mitochondrial damage and relevant mechanism research.(1)S.cerevisiae BY4743 cells were irradiated with different doses(30,60,90,120,150,180 Gy)carbon ion beam with the energy of 80 MeV/u and the dose rate of 20 Gy/min.The yeast cells death rate was determined by plate colony counting method and survival curve was drawn.The semi-lethal dose is 60 Gy.Based on the TTC plate experiment,10 mitochondrial-damaged yeast mutants were screened.The mutants screening rate reached the highest at 150 Gy.The growth curve of yeast after carbon ion beam irradiation can divide the dose into three levels including low,medium and high.The high dose(180 Gy)has a strong lethal effect on yeast.(2)The proliferation rate and respiratory intensity of the 10 mutants were determined.It was found that the mutants with a proliferation rate lower than 50% of the control accounted for 80%,and the mutants with a respiratory intensity lower than 50% of the control accounted for 90%.According to this results,five mutants with obvious mitochondria damage were rescreened,namely ResD-1,ResD-2,ResD-3,ResD-4,and ResD-5,respectively.The mitochondrial transmembrane potential of the mutant cells were detected by flow cytometry after Rhodamine 123(Rh123)staining.The results showed that the mitochondrial transmembrane potential of all mutants were reduced about 10 times compared with the control.Based on the genome sequencing data,the mitochondrial genomes coverage depth were compared.It was found that the mitochondrial genomes showed large fragment deletion in mutants.Combined with the nuclear genome results,it can concluded that mitochondrial DNA damage is one of the crucial reasons for phenotypic changes.(3)The lipid components of the five mutants were detected by ultra-performance liquid chromatography-electrospray ionization-mass spectrometry(UHPLC-ESI-MS)combined with multivariate variables and univariate data analysis methods.The quality control(QC)sample proves that the analytical detection system used in this study is stable and reliable.The results can be used for the next analysis.Based on UHPLCESI-MS method,648 kind of lipid molecules belong to 25 lipid subclasses were identified in the control and mutant groups.140 kind of these lipid molecules accounting for 21.6% of total lipids are neutral lipid TG and the rest are phospholipids.The overall sample PCA results accurately differentiated between control and mutant.Based on the distribution of the samples on the PCA score map,ResD-1 and ResD-4 were identified as the main subjects(4)Orthogonal partial least squares discriminant analysis(OPLS-DA)was performed on the 648 kind of lipid molecules.The results show that OPLS-DA can accurately distinguish ResD-1,ResD-4 and Control.By combined analysis of variable weight Value(VIP),variation multiple(FC)and differential(p),10 subtypes including DG(16:0/18:1),LPC(16:0),LPE(14:0),LPI(16:0),PA(16:1/18:1),PC(24:0),PE(16:0/10:0),PI(18:0/18:1),PS(16:0/16:1),TG(16:0/10:0/16:1)and 51 kind of significant differential lipid molecules were screened in ResD-1.And 12 subclasses including DG(16:0/18:1),LPC(18:1),LPE(16:0),LPI(18:1),LPS(18:1),PA(16:0/18:1),PC(31:1),PE(16:0/10:0),PI(15:0/18:1),PS(16:0/16:1),SQDG(37:4),TG(15:0/16:0/18:1)and 54 kind of significant differential lipid molecules were screened in ResD-4.Significant differences in lipid molecular relative content analysis showed that in ResD-1/Control,the content of DG,TG,LPI,PA and PI were increased,the content of LPC,LPE,PC,PE and PS were decreased.In ResD-4,the content of DG,TG,SQDG,PA,PC,PI,LPC,LPE and LPI were increased,and the content of PE,PS and LPS were decreased.Significant differences in lipid clustering analysis can be used to screen for lipid markers and to facilitate the search for similar lipid metabolic pathways.Based on the above results,metabolic pathways of lipids such as DG,PA,PI,and PS were constructed.This study indicated that S.cerevisiae may respond to mitochondrial damage caused by heavy ion beam irradiation by regulating the content of lipid components.The lipid molecules with significant difference expression level may be potential markers for S.cerevisiae mitochondrial damage induced by heavy ion beam irradiation.(5)Based on the RNA-Seq technique,transcriptome of 9 samples of Control,ResD-1 and ResD-4 was sequenced,and 6252 genes were obtained.Quality detection and comparison efficiency statistics were obtained for the sequencing data,which proved that both the sequencing data and the reference genome can meet the analysis requirements.The results of SNP analysis indicated that the SNPs occurring in the mutants are background mutations,and the yeast mitochondrial damage induced by heavy ion beam irradiation is the main cause of the mutation.Combined with the fold change(FC)and false discovery rate(FDR),622 and 1011 differentially expressed genes were screened in ResD-1 and ResD-4 vs Control,respectively.ResD-1,ResD-4 vs Control commented that the DEG of the GO secondary node was 445 and 741,respectively.The DEG of the biological process,exceeding 40%,was the highest.Compared with the COG database,the total number of DEGs reported by ResD-1,ResD-4 vs Control is 339 and 555 respectively.Compared with the KOG database,the total number of DEGs annotated is 439 and 769,respectively.The results of COG and KOG annotations show that the most classified categories of DEG annotations are “general functions prediction only”.In ResD-1 vs Control,COG and KOG accounted for 2.65% and 5.24% of lipid transport and metabolism,respectively;in ResD-4 vs Control,COG and KOG annotated ratios were 1.44% and 3.77%,respectively.The results of KEGG pathway analysis showed that ResD-1,ResD-4 vs Control wereannotated to 160 and 233 DEG,respectively.The carbon metabolism,amino acid biosynthesis and pyrimidine metabolic pathways were the most detailed DEG.There are 9 pathways which divided into 5 aspect including fatty acid biosynthesis and degradation,glycerides,glycerophospholipids,sphingolipids and sterols related to lipid metabolism.There are 20 and 23 DEGs,accounting for 12.5% and 9.87% of the total DEG of KEGG related to lipid metabolism in ResD-1 and ResD-4 vs Control,have changed.And the above DEGs are mainly down-regulated.(6)Based on the DEG annotated in the lipid metabolism pathway,16 key genes such as YIL009 W,YOR374W,YBR029 C,YBR042C and YBR183 W were selected for qRT-PCR verification of the expression levels.It was showed that data can correspond well with RNA-Seq results.These results indicate that the RNA-seq results can accurately reflect the differential expression of lipid metabolite-related genes in the mutants. |
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