Analysis Of Mitochondrial DNA Barcode And Expression Of HSP70 Under ¹³⁷Cs-? Radiation Stress In Laboratory Red Crucian Carp

Objective:1.In order to further improve the genetic background of Laboratory red crucian carp and establish the mitochondrial molecular biomarkers of Laboratory red crucian carp,it was proposed to carry out mitochondrial DNA barcode analysis of Laboratory red crucian carp.2.To promote the application research of Laboratory red crucian carp in the biological effects of nuclear radiation,the study discussed the influence of ¹³⁷Cs radiation on the expression of HSP70 in Laboratory red crucian carp.Methods:1.The primers were designed to amplify COI,COIII,Cytb and D-loop genes of Laboratory red crucian carp C1HD,goldfish,Goldfish-like fish,Red crucian carp-like fish,Carassius auratus L and Carassius cuvieri.The target band is recovered,transformed and sequenced to determine sequence results.Using Cyprinus carpio as the outer group,the sequence characteristics,intraspecies genetic distances,interspecies genetic distances,and Molecular Phylogenetic relationship were analyzed combined with the sequence downloaded from NCBI.2.Mitochondrial genome sequences of the Laboratory red crucian carp,Goldfish-like fish,Red crucian carp-like fish and Goldfish were obtained by sequencing,and the structural characteristics,base composition and codon usage frequency of the whole mitochondrial genome sequence in Laboratory red crucian carp were analyzed.Finally,the phylogenetic tree was constructed combined with the mitochondrial genome sequence of Carassius downloaded from NCBI.3.According to the LD_50/30 of ¹³⁷Cs radiation from Laboratory red crucian carp,4.1Gy,6.1Gy,8.2Gy group and control group were designed.After 1 day of ¹³⁷Cs-?irradiation,the liver and kidney tissues of the Laboratory red crucian carp were extracted and the expression of HSP70protein was detected by Western blot.Results:1.The results of base composition analysis showed that the A+T content were higher than G+C in COI,COIII,Cytb and D-loop genes of the studied fish.Intraspecies genetic distances based on COI,COIII,Cytb and D-loop were 5.21%,3.8%,6.1%,5.5%,while interspecies genetic distances were 0.18%,0.1%,0.13%,0.21%,respectively.The average interspecies genetic distance was the average intraspecies genetic distance 28.6,38,46.9 and 26.1 times.The average interspecies genetic distance was consistent with more than 10 times of the intra-species genetic variation threshold.The phylogenetic tree clustering results based on COI,COIII,Cytb and D-loop gene sequences were basically the same.The phylogenetic tree can be divided into two branches.The outgroup?carassius cuvieri?formed one branch,and all fish from carassius was gathered into one.The Laboratory red crucian carp and Goldfish were gathered together,and carassius auratus grandoculis and carassius auratus langsdorfii were gathered together,and Other fish was a monophyletic group separately,which is consistent with the results of taxonomy.2.The mitochondrial genome sequences of the Laboratory red crucian carp,Goldfish-like fish,Red crucian carp-like fish and goldfish measured 16581 bp.The mitochondrial genome contained 13protein-coding genes,22 tRNAs,2 rRNAs,and 2 non-coding regions.Mitochondrial genes were arranged closely with the short spacer or overlap of contiguous genes.In terms of base composition,except for the OL region,all parts of the mitochondrial genome showed bias toward AT.The third codon also showed bias toward A and T.From the evolutionary relationship,the phylogenetic tree constructed based on the mitochondrial genome and 13 coding genes was consistent with the topology of phylogenetic tree constructed by a single gene.3.Nuclear radiation stress can induce the increase of HSP70expression in liver and kidney of the Laboratory red crucian carp.Conclusion:1 The experimental mitochondrial COI,COIII,Cytb,D-loop gene and mitochondrial genome have germplasm conservation equal to species-specific nucleotide sequences,and A+T content is higher than G+C.2 COI,COIII,Cytb,D-loop gene and mitochondrial genome can be used to distinguish the molecular markers of Laboratory red crucian carps and other fish from carassius,but not for line authentication.3 A certain dose of ¹³⁷Cs irradiation can increase HSP70 expression in the liver and kidney of Laboratory red crucian carp.