Expression Of LINE1 In Zebrafish Under Cold Acclimation

Transposons are found in the genome of eukaryotes and are called junk genes.To date,the three retrotransposons are still active: LINE1(L1),Alu and SVA elements.L1 retrotransposon is a hopping gene with a large number of copies in the genome,which accounts for 17% of the human genome and more than 10% of the zebrafish genome.They use the "copy-and-paste" mechanism to propagate through the entire genome through RNA intermediates,a process called reverse transcription.L1 s are active during germline and embryogenesis,but they are epigeneticly suppressed in somatic cells.L1 with reverse transcriptional transcription activity is about 6 kb long and contains a 5’ untranslated region(UTR),two open reading frames(ORF1 and ORF2),and a 3’UTR ending with a poly(A)tail.The 5’UTR contains a CpG island and an internal promoter that drives transcription of the full-length L1 transcript;both ORF1 and ORF2 are required for L1 transposition,and the function of ORF1 remains unclear,but it has been shown to have nucleic acid chaperone activity,Involved in the formation of L1 ribonucleoprotein particles(RNP),ORF2 is highly conserved in mammals and contains endonuclease and reverse transcriptase domains;3’UTR shows very little conservation between species.Reversal blocks lead to genomic changes,most of which have negative effects on the genome,such as changing the structure of the genome,affecting gene expression,and changing the way genes are regulated.Therefore,the host gene will be methylated by promoter regions or small RNA interference.Suppress transposition.However,more and more studies have shown that transposable elements and genomes are mutually beneficial.Reversal of transposons can lead to the generation of new genes,which is positive for species diversity and species evolution.When faced with environmental factors,the genome undergoes its own evolution to induce transposition of transposons,allowing organisms to adapt to environmental changes.Fish as a temperature-changing animal,water temperature changes affect its physiology and behavior to a large extent.The Antarctic continent has experienced a long cooling,the water temperature is below 0 °C all year round,how the dominant species in the Antarctic fish Notothenioids in the Antarctic circle survives in extreme cold,and resisting the harsh environment is an important issue.Studies have found that the genome of the Antarctic fish in the low temperature is relatively large compared to the genome of fish outside the Antarctic.These results also indicate that in cold environments,Antarctic fish evolved a way to resist cold and produced new genes,and the production of these new genes may be related to the expansion of LINE.Much of the knowledge of the effects of the environment on the physiology and evolution of vertebrates comes from the study of fish,which have most physiological mechanisms with humans.The physical and physiological relationships with the aquatic environment give high sensitivity to challenge the environment and have important advantages in defining bio-environmental interfaces and response to temperature adaptation.The zebrafish(Danio rerio)is a tropical fish.Due to its short age,many offspring,clear genetic background,it is easy to carry out experimental operations,easy to breed in the laboratory,and is similar to human genes 87%,thus serving as a model organism.In biology.In this experiment,zebrafish was used as an experimental material to study the function of LINE1 in low temperature adaptation.In order to study whether large-scale amplification of transposon in long-term low temperature environment is related to cold adaptation.The cells were cultured at 18 ° C,and it was found that the cells were stagnation after 5 days of treatment in a low temperature environment of 18 ° C,and gradually recovered after about 20 days,although it was still slow compared to 28 ° C.In the previous study,it was found that the L1 methylation level was changed in the low temperature environment.The methylation level of 5d L1 in ZF4 cells was increased at 18 °C,and the methylation level of ZF4 cells was decreased at 30 °C.The level of L1 is regulated by the level of methylation,and it is estimated whether the low temperature affects the expression amplification of L1 by affecting the 5’UTR activity,which may be one of the ways in which the organism responds to the external low temperature environment and finds The shizothoracine fish living under anoxic conditions also showed a significant increase in the expression of LINE in the genome.These conclusions indicate that in extreme environments,the genome of the organism regulates LINE amplification,increases genomic diversity,and responds to changes in the external environment.In this experiment,the zebrafish(Danio rerio)embryonic fibroblast ZF4 was treated with low temperature for different lengths of time(18°C for 5d and 18°C for 30d),and the zebrafish adult fish were also treated with low temperature for different lengths of time(10°C).3h 6h 1d 3d 5d),the mRNA level of L1 was detected by RT-qPCR;the promoter region of zebrafish L1 gene was cloned,and the biological activity of 5’UTR in cells was verified in ZF4 cells by Luciferase dual fluorescence reporter system.The 5’UTR activity was monitored at low temperature(18 °C 3d).The results showed:(1)In the cells,the L1 mRNA level was decreased in the short-term low temperature treatment,while in the long-term low temperature treatment,the mRNA level of L1 was greatly increased.(2)In adult fish,the L1 mRNA level was decreased under short-term low temperature treatment,and the L1 mRNA level was increased in long-term low temperature treatment.(3)The 5’UTR was found to be biologically active in ZF4 cells.(4)It was found that the reporter gene signal was weakened at low temperature(3 °C for 3d),which indirectly indicated that the L1 promoter activity was weakened.It is speculated that L1 has a positive effect on the adaptation of organisms to external low-temperature environmental stress.The transposition of transposons in organisms increases in cold environments,and the diversity of matrix groups increases,which adapts to environmental stress.In summary,this study through the low temperature treatment of zebrafish cells and zebrafish adult fish for different lengths of time,study the mRNA level of L1,and the change of activity in the 5’UTR region at low temperature to lay a better understanding of the cold adaptation mechanism.The foundation.