Establishment Of ⁶⁰Co-?-ray Radiation Model Of Aurita Coerulea Polyps And Screening Of Related Genes

BackgroundWith the wide application of nuclear reaction technology,medical radiation technology and the rapid development of nuclear industry,various health problems caused by radioactive exposure have attracted more and more attention.In the nuclear polluted ocean,marine organisms can absorb radioactive waste through contaminated food and water,so that they are directly exposed to nuclear radiation in vitro and in vivo.Through direct ionization and chemical action,as well as the indirect effect of the generated free radicals?OH and?H,which attack and destroy nucleic acids,proteins,enzymes and other biological macromolecules,causing serious damage to marine organisms and even threaten life.Jellyfish is one of the most primitive group in the multicellular marine organisms,and canonically have both asexual/sessile and sexual/planktonic stages in their metamorphosis life cycle,the drifting medusae swims freely in the ocean and the benthic polyps attached to the seabed.Compared with the medusae,the polyps can maintain the quantity in this stage by budding,and induce metamorphosis to produce jellyfish.Futhermore,the mature medusae can release sperm and combine with eggs to form fertilized eggs to achieve further increase of population through sexual reproduction.Therefore,the polyp plays a more important role in maintaining and expanding the population of jellyfish.Through 500-600 million years of successful biological evolution,the jellyfish has acquired strong reproductive plasticity and environmental adaptability,which makes it occupies diverse habitats,ranging from tropical seas to subarctic regions,as well as from the open ocean to brackish estuaries.A.coerulea is the most common and widely distributed representative jellyfish around the world.In addition,A.coerulea polyp is the first one to achieve artificial breeding and reproduction,the earliest genome analysis and the most in-depth study of jellyfish species,from the emergence of polyp to the metamorphosis of jellyfish,etc.It can be said that the A.coerulea is a representative of low-level marine multicellular invertebrates,especially its polyp is gradually growing into an ideal model organism to study the marine environment adaptability of marine life.When nuclear pollution is faced,such as nuclear leakage accident in nuclear power plant,due to radioactive waste deposition,benthic A.coerula polyp in the sea near nuclear power plant will be more polluted by radiation than drifting jellyfish,which may lead to abnormal metamorphosis process and affect the quantity and quality of jellyfish populations.It may bring further secondary marine nuclear pollution with the freely swimmed medusae.In terms of nuclear radiation related medical research,the damage effect of nuclear radiation induced A.coerulea polyp is analyzed as the marine low-level model animal,and the difference between them and mammals is analyzed.Therefore,it will provide important reference for the new mechanism,new target and new intervention strategy research of nuclear radiation damage from the evolutionary perspective.Therefore,based on the self-built feeding and observation system of A.coerulea polyp in our laboratory,the damage effect of ⁶⁰Co-?radiation on the polyp and its symbiotic microorganisms by ⁶⁰Co-?radiation were studied in the thesis.It is of great significance to affect the marine nuclear radiation pollution on the marine low-level multicellular organisms,explore new mechanism,target and intervention strategy of nuclear radiation damage by using marine low multicellular organisms as reference.Contents1.Identification of the A.coerulea and its polyp,and construction of the laboratory micro feeding system of A.coerulea polyp.2.Construction and evaluation of ⁶⁰Co-?radiation injury model of A.coerulea polyp.The key molecules and pathways related to radiation injury of A.coerulea polyp were screened,and the effects of radiation injury related molecules of A.coerulea polyp on radiation injury of mammalian 293T cells were analyzed based on the transcriptome.3.The effect of ⁶⁰Co-?radiation on the symbiotic bacteria of A.coerulea polyps.Methods1.Identification of A.coerulea and its polyp and construction of laboratory micro feeding system of polyp(1)Species identification and analysis of the A.coerulea and its polypAll the annotated gene sequences of cytochrome c oxidase subunit I(COI)were screened from the transcriptome of the A.coerulea and its polyp.The Blastn and Blastx were compared with the data in NCBI nucleic acid database,and the species information with the greatest similarity to the sequence of the tested species was obtained,which was the identification result.Bioedit was used to compare the two selected sequences with the nucleic acid and protein sequences with the highest similarity.Then,MEGA7 were used to build the evolutionary tree.(2)Establishment of micro feeding system for A.coerulea polypsThe polyps used in this study were cultured in artificial seawater with 3%salinity and biochemical incubator at 20?using petri dish and six well plate.It mainly includes seven steps:artificial sea water preparation,bait preparation,transfer,attachment,feeding,artificial sea water replacement and subculture.2.Damage effects of ⁶⁰Co-?radiation on A.coerulea polyps and screening of related genes(1)The establishment and evaluation of ⁶⁰Co-?radiation damage model of A.coerulea polypsA dose-response model of radiation damage was established by 0?1000 Gy ⁶⁰Co-?radiation.In addition,the biological effects of radiation damage,such as the stretching of the tentacles,the activities of superoxide dismutase(SOD),glutathione peroxidase(GSH-Px),malondialdehyde(MDA),catalase(CAT),total antioxidant capacity(T-AOC)and pathological examination were observed to complete evaluation.(2)Based on transcriptome analysis and screening of key molecules and pathways related to radiation injuryThe total RNA was extracted from the A.coerulea polyp samples after 900 Gy ⁶⁰Co-?radiation and the control group,and the Illumina hiseq4000 sequencer was used for transcriptome sequencing and annotation analysis inc Hangzhou Lianchuan biological company.Futhermore,the differentially expressed genes were annotated by NR and Swissprot,enriched by GO and KEGG pathways,and the potential key differential molecules were screened from the transcriptome.The damage related genes were compared with corresponding human homologous genes to find out the difference fragments,core fragments,and the 3D structure anaylsis of these molecules were performed using SWISS-MODEL,RCSB PDB,and NCBI blast,etc.Therefore,we can preliminarily understand the possible biological functions of the selected genes through the results collected in the database.3.The effects of radiation damage related molecules of the A.coerulea polyp on radiation damage of mammalian 293T cellsRT q PCR was first used to verify the accuracy of transcriptome sequencing.After the full length of key molecules was amplified by PCR,the overexpression plasmid containing key molecules related to radiation injury was constructed by molecular biological methods.The correctness of the amplified sequence was confirmed by bacterial fluid PCR and Sanger sequencing.Then,the target plasmid was extracted and transfected into mammalian 293T cells by Lipofectamine after amplification by E.coli.The expression was verified by Green fluorescent labeling,PCR and Western Blot.Finally,in order to explore the effect of target molecules on radiation injury of mammalian cells,the CCK8 method was used to detect cell viability and flow cytometry was used to detect cell apoptosis.3.Effects of ⁶⁰Co-?radiation on the symbiotic bacteria of A.coerulea polypDNA was extracted and 16S r RNA gene was sequenced from the four parts of the umbrella,tentacles,oral arm,gastric cavity and A.coerulea polyps before radiation and at different time points(within 2 hours after radiation,1,3 and 5 day after radiation)after 900Gy ⁶⁰Co-?radiation.QIIME and R package(v3.2.0)were used for sequence data analysis.PICRUSt 2(KEGG pathway database)based on high quality sequences was used to predict microbial function.Through the above methods,the symbiotic bacteria of A.coerulea and its polyps were compared and analyzed,as well as the effect of ⁶⁰Co-?radiation on the symbiotic bacteria of polyps.Results1.Identification of A.coerulea and its polyp and construction of laboratory micro feeding system of polyp(1)Species identification and analysis of the A.coerulea and its polypWe observed the metamorphosis and development process of the jellyfish in the laboratory,mainly including four development stages:polyps,strobila,ephyrea,and medusae,which is consistent with the metamorphosis and development process of the traditional Aurelia sp.The appearance characteristics of the laboratory induced adult medusae was consistent with that of the Aurelia sp,which can be preliminarily judged that the animals we studied belong to Aurelia.At the molecular level,the conserved COI genes,TRINITY?DN16912?c0?g3 and TRINITY?DN11321?c3?g4,were successfully screened from the transcriptome of jellyfish and its polyp.The two sequences were compared with the NR database of online tools Blastn and Blastx.The results showed that the COI genes closest to the two sequences were all from species A.coerulea,and the similarity was 98.90%,98.95%and 99.03%,99.21%respectively.Therefore,through the analysis of metamorphosis,morphology and gene molecular level,we finally confirmed that jellyfish we used is A.coerulea,a subspecies of Aurelia sp.(2)Establishment of micro feeding system for A.coerulea polypWe have successfully constructed a micro feeding system of A.coerulea polyps in the laboratory,including the preparation of artificial seawater with a salinity of 2.8?3.2%,the preparation of A.coerulea polyps bait(hatching of shrimp eggs),the transfer of A.coerulea polyps from corrugated plate to six well plate(5?10 per well)or Petri dish(20?30 per plate)covering,recording the planting date and putting it into 20?biochemical incubator,attaching A.coerulea polyps to the bottom of plate/dish without feeding or changing water within one week,feeding A.coerulea polyps every two days after attachment and replacing fresh artificial seawater after feeding for 2 hours,and subculturing polyp after full growth.At present,the method has applied for a domestic invention patent and entered the substantive examination stage.2.Damage effects of ⁶⁰Co-?radiation on A.coerulea polyps and screening of related genes(1)The A.coerulea polyps showed obvious tolerance to ⁶⁰Co-?radiationInterestingly,the activity of A.coerulea polyps showed a dose-dependent inhibitory effect,and it could return to normal within 5 days after undergoing 0?900 Gy ⁶⁰Co-?radiation,while the polyps gradually died within a week when the dose was more than 900 Gy,and the mortality rate reached 100%when the dose was more than 1000 Gy.Therefore,the A.coerulea polyps showed obvious radiation tolerance and the ⁶⁰Co-?radiation dose damage effect model was established with 900 Gy(9 Gy/min dose rate).HE staining results showed that,the main microstructure of polyps did not change significantly,although they had obvious symptoms and biochemical changes at 900 Gy.The activity of oxidoreductase system showed that SOD,CAT,GSH-Px and T-AOC of polyps decreased first and then increased within 24 h after ⁶⁰Co-?radiation,which may be related to the early elimination of ROS and the late increase of feedback.(2)Screening key molecules and pathways related to radiation injury based on transcriptome analysisA total of 52,759,915 reads were obtained by Illumina sequencing,and 42,684,781(96.98%)clean reads were obtained after removing the linker and low-quality sequence.A total of 72,678 transcripts were assembled by Trinity software.Compared with the control group,2,748 genes were up-regulated and 3,361 genes were down-regulated in the radiation group.In addition,KEGG pathway enrichment and GO annotation analysis showed that different from TLR,NF-?B and MAPK signaling pathways common in mammalian radiation injury,the important pathways such as endocytosis,mitochondrial autophagy,protein processing and synthesis,oxidative phosphorylation and maintaining normal life activities were up-regulated in radiated polyps.In addition,DNA excision,repair,transcription regulation,nucleic acid binding and other pathways related to DNA and RNA synthesis were down regulated.SQSTM1 and HSPA8 changed the most after radiation among the annotated molecules,with the expression levels of 2,538.01 and 986.18,and log₂FC of 6.52 and 6.11,respectively.(3)The effects of radiation injury related molecules of A.coerulea polyp on radiation injury of mammalian cellsBased on the 46 differentially expressed genes related to radiation injury,the up-regulated top 10 genes after radiation were constructed successfully,including Adtrack-a1-Flag,Adtrack-SQSTM1-Flag,Adtrack-Maf A-Flag,Adtrack-a4-Flag,and Adtrack-a10-Flag.Then,the plasmid was extracted and transfected into mammalian 293T cells by Lipofectamine after amplification by E.coli.The expression of Adtrack-a1-Flag,Adtrack-Maf A-Flag,and Adtrack-a10-Flag were verified by Green fluorescent labeling,PCR and Western Blot.Further study showed that the overexpression of Maf A and a10 could reduce the late apoptosis rate of 293T cells to a certain extent.3.Effects of ⁶⁰Co-?radiation on the symbiotic bacteria of A.coerulea polypOur results suggested that Proteobacteria(76.19±3.24%),Tenericutes(12.93±3.20%)and Firmicutes(8.33±1.06%)are most abundant in medusae,while Proteobacteria(29.49±2.29%),Firmicutes(46.25±5.59%),and Bacteroidetes(20.16±2.65%)are the top three phyla in polyps.After ⁶⁰Co-?radiation,the proportion of Proteobacteria increased from 29.49±2.29%to 59.40±3.09%over 5 days,while that of Firmicutes decreased from 46.25±5.59%to 13.58±3.74%.At the class level,Gammaproteobacteria continually increased during the 5 days after radiation exposure,whereas Bacilli declined,followed by partial recovery,and Alphaproteobacteria and Flavobacteriia remained almost unchanged.Conclusion1.We have successfully established a stable laboratory micro feeding system for the A.coerulea polyps.2.The A.coerulea polyp has a significant dose-response relationship to ⁶⁰Co-?radiation,showing a tolerance to ⁶⁰Co-?radiation.A stable dose-response model of radiation damage to the polyp was successfully established with 900 Gy(9 Gy/min)⁶⁰Co-?radiation.3.The antioxidant enzymes(SOD,CAT,GSH-Px and T-AOC)of the A.coerulea polyps first decreased and then recovered within 24 h after radiation,indicating that the ⁶⁰Co-?ray tolerance of the A.coerulea polyps may be related to the strong oxidative defense system.4.Different from the classical TLR,NF-?B and MAPK signaling pathways in response to radiation injury in mammals,the up-regulated GO and KEGG signaling pathways were mainly related to endocytosis,mitochondrial autophagy,protein processing and synthesis,oxidative phosphorylation and other important pathways in maintaining normal life activities.It was suggested that there was a significant difference in the response to radiation injury between A.coerulea polyps and mammals,which provides a new reference for the discovery of new mechanisms and targets of radiation injury in mammals.5.The different genes Maf A and a10 related to the damage of A.coerulea polyps can reduce the proportion of late apoptosis of 293T cells after radiation,suggesting that the effector molecules of A.coerulea polyp after radiation have the potential of radiation protection to a certain extent.6.The dynamic changes and redistribution process of the symbiotic and epiphytic microorganisms in the A.coerulea polyps after ⁶⁰Co-?radiation may be an important mechanism for the adaptation of the polyps to the external environment including radiation,and contributed to the tolerance and adaptation of the lower marine multicellular invertebrates such as the polyps to the external environment.