The Comparative Study On Response Mechanism Of Platycodon Grandiflorus To Heavy Ion Beam And X-ray Radiation

Platycodon grandiflorus（P.grandiflorus）is a perennial herb.The root,commonly used Chinese herbal medicine,plays an important role in the prevention of the novel coronavirus infection.P.grandiflorus also has edible and ornamental value,and broad market prospects.However,the problem of variety degradation occurred in the long-term cultivation process.Germplasm innovation and variety improvement are urgently needed.Physical mutagens are widely used in plant germplasm innovation,and many new varieties have been obtained in a variety of crops and ornamental plants.Most medicinal plants are perennial,self-incompatibility,long selection and breeding cycle,unclear genetic background,and difficult gene editing,that restrict the breeding progress of medicinal plants.Radiation mutagenesis breeding is an effective method to create genetic variation,which has no special demands on species,sample physiological state and genetic background.This method can greatly promote the development for medicinal plant germplasm resources.X-ray is conventional photon radiation.In contrast,the linear energy transfer（LET）and relative biological effectiveness of heavy ion beam radiation are high.It shows the characteristics of high mutation rate,wide mutation spectrum and relatively short breeding cycle in mutation breeding.However,the effects of different LET radiation on the phenotypic,physiological and molecular levels of medicinal plants have not been systematically analyzed.In this study,the medicinal plant P.grandiflorus were used as radiation materials to explore the effects of heavy ion beam and X-ray radiation on the germination rate,the survival rate,the cell membrane damage,the antioxidant enzyme activity,the gene expression,the protein expression,the growth and development,the secondary metabolites of P.grandiflorus.In addition,the peroxidase classⅢ（PERs）gene family of P.grandiflorus was identified by bioinformatics method,and the expression pattern of PERs gene after different radiation was analyzed.The following results were obtained:1.The dry seeds of P.grandiflorus were irradiated by heavy ion beam and X-ray,and the germination rate,the survival rate,the malondialdehyde content and the antioxidant enzyme activity after irradiation were measured.The median lethal doses(LD₅₀)of heavy ion beam and X-ray radiation are 175 Gy and 264 Gy,respectively.The malondialdehyde content of P.grandiflorus is significantly increased after 100Gy heavy ion beam irradiation,while X-ray irradiation has no effects on the malondialdehyde content.Two kinds of radiation has no significant effect on catalase activity.After heavy ion beam irradiation,peroxidase activity increased slightly,and superoxide dismutase activity did not change.The activities of peroxidase and superoxide dismutase decreased significantly after X-ray irradiation.A total of 133differentially expressed genes（DEGs）were induced by 100 Gy heavy ion beam irradiation,and X-ray irradiation resulted in more DEGs（369）.DEGs induced by heavy ion beam and X-ray irradiation were enriched in different metabolic pathways:DEGs after heavy ion beam irradiation were mainly enriched in protein kinase activity,hydrogen peroxide,reactive oxygen species,and stress response pathways.X-ray irradiation stimulated the gene expression in oxidoreductase,hydrolase activity and carbohydrate metabolism pathway.KEGG enrichment results showed that heavy ion beam irradiation activated the gene expression of galactose,amino sugar and nucleotide sugar metabolism,zeatin biosynthesis.After X-ray irradiation,the gene expression levels of plant secondary metabolism,monoterpenoid biosynthesis,starch and sucrose metabolism and Mitogen-activated protein kinase（MAPK）signaling pathway were significantly increased.After heavy ion beam irradiation,12 MAPK pathway genes were differentially expressed,and X-ray induced significant changes in the expression of 29 MAPK signal transduction genes,indicating that X-ray irradiation induced more complex MAPK regulation.2.P.grandiflorus seedlings were used as radiation materials to explore the effects of two kinds of ionizing radiation on gene and protein expression patterns,plant growth and secondary metabolites.The LD₅₀ of heavy ion beam and X-ray radiation are 24.6 Gy and 35.8 Gy,respectively.The leaf area decreased with the increase of absorbed dose.At the same dose,the inhibitory effect of heavy ion beam on the growth of P.grandiflorus leaves was stronger than that of X-ray.The12-day-old Platycodon grandiflorum seedlings were irradiated with the same effect dose of radiation（15 Gy heavy ion beam and 20 Gy X-ray）.RNA-Seq and proteomics were used to analyze the regulation of gene transcription and protein expression induced by two kinds of irradiation.The number of DEGs induced by heavy ion beam radiation was less than that of X-ray group,but heavy ion beam radiation induced more differential proteins.Both heavy ion beam and X-ray irradiation activated genes of RNA silencing,double-strand break repair,and cell catabolic process.DNA replication and cell cycle related genes were down-regulated.The genes of cell wall and external encapsulating structure were up-regulated after heavy ion beam irradiation.The gene expression of protein folding and glucan biosynthesis increased after X-ray radiation.Protein enrichment analysis showed that heavy ion beam irradiation resulted in differential protein enriched in photosynthesis and secondary metabolite biosynthesis pathways,while X-ray irradiation resulted in differential protein enriched in glyoxylate and dicarboxylate metabolism and carbon metabolism.After heavy ion beam and X-ray irradiation,the genes of antioxidant system and terpenoid and polyketide metabolic pathways showed different expression patterns.X-ray irradiation leads to the enrichment of precise homologous recombination repair pathways,while non-homologous end-joining pathways are enriched after heavy ion beam irradiation,which increases the probability of mutation.Beside,5-15 Gy heavy ion beam and 10-30 Gy X-rays promoted the early flowering of P.grandiflorus,and caused variations in petal and stigma number.Heavy ion beam lead to mutant of male sterility and sepal color.3.In order to explore the role of PERs gene family in the response of P.grandiflorus to different quality ionizing radiation,we identified the P.grandiflorus PERs（Pg PERs）family members and analyzed the expression regulation of Pg PERs gene after heavy ion beam and X-ray radiation treatment.53 Pg PERs were obtained by sequence alignment,domain identification and protein conserved motif analysis.The sequence length of proteins are mainly distributed between 258 and 621 amino acids,and alkaline protein are the majority.The analysis of promoter regulatory elements showed that Pg PERs have many cis-acting elements related to growth and development,plant hormone and stress response.According to the evolutionary analysis,the Pg PERs are mainly divided into five clusters,and have 29 pairs of collinearity with At PERs in Arabidopsis thaliana.Heavy ion beam resulted in up-regulation of six Pg PERs,while X-ray induced down-regulation of eight Pg PERs.These DEGs encode proteins withα-helix and random coil as the main secondary structure.The tertiary structures is highly similar and contain transmembrane helices.In conclusion,the LD₅₀ of heavy ion beam radiation was lower than that of X-ray.Heavy ion beam induced more types of phenotypic variation,and the relative biological effectiveness was higher than that of X-ray.Heavy ion beam radiation induced fewer differential genes and more differential proteins.The two radiations induced the response of genes and proteins in different metabolic pathways of P.grandiflorus.The main repair pathway after heavy ion beam radiation is non-homologous end-joining,which is easy to introduce errors and produce mutations,which is the reason for the high mutation frequency of heavy ion beam.These results provide a theoretical basis for radiation breeding of P.grandiflorus and other medicinal plants.