Effects Of Anti-Freeze Gene Ld4 Of Polar Fish On The Formation Of Stress Granules In ZF4 Cells Under Cold Stress

Long evolution in the process of the earth,climate changing,the polar sea water temperature changes gradually from 15 ? to-1.9 ?,led to the deaths of many polar fish,and some of the polar ocean fish in order to adapt to the continuous low temperature,gradually evolved the mechanism of low temperature resistant to cold,including the generation of antifreeze protein(AFP)is a kind of typical polar fish mechanisms of adaptive evolution.Scholander and DeVries were the first scientists to discover that some fish in polar oceans can survive in sub-zero cold water.Later,studies found that polar fish had a class of cold-resistant proteins called AFPs.Currently found in fish,5 kinds of antifreeze proteins,AFP ?,AFP ?,AFP ?,AFP ? AFGPs and protein sequences between the several kinds of AFPS features,spatial structure and evolutionary origin in huge differences between different species,such as antifreeze function may be related to its sequence and structure specificity.Antifreeze proteins work by inhibiting the formation of ice crystals in polar fish.Antarctic fish Lycodichthys dearborni(LD),the body contains rich AFP ?,our laboratory from the Antarctic fish of AFP gene ld12 ? polymer cDNA cloning have been AFP ? four polymers,proteins build LD4 plasmid.Cold stress can lead to physiological dysfunction of fish,damage to the body and even death.The cells will produce various adaptive changes to cope with this cold stress to facilitate survival.Under various stresses,the production of stress granules was found in mammalian cells and yeast cells.The production of stress granules(SG)is a self-protective defense mechanism of cells.It inhibits the translation of mRNA in cells when exposed to environmental stress(such as heat shock,oxidative stimulation,viral infection,etc.).A protein-containing dense particle containing a translation initiation factor,untranslated mRNA,and G3BP1,TIA-1,etc.is formed.Such particles have been confirmed in yeast,plant,mammalian cells.The study of stress granules under cold stress mainly focuses on mammalian cell models,which are rarely reported in fish cells.Based on their functional similarity,when studying the function of ld4 in laboratory,we found in HeLa cells that the expression of ld4 can reduce the formation of stress granules in cells under cold stress.This provides a basis for us to study the relationship between the two in zebrafish embryonic fibroblasts(ZF4).This experiment will first explore the formation of stress granules in ZF4 cells under cold stress.We set the temperature gradients of 18°C,13°C and 10°C as the cold stress.After different time of low temperature treatment,we used cellular immunofluorescence experiments and RT-qPCR experiments to investigate the formation of stress granules and the depolymerization of SG during stress recovery.The results are as follows:1)ZF4 cells under cold stress,compared with cells cultured at 28°C,whether at 18°C,13°C or 10°C low temperature treatment,by immunofluorescence experiments,ZF4 cells will aggregate under cold stress at different temperatures.Stress granules were formed,and the stress granules were most formed when treated at 10°C for 1 h,13°C for 4 h,and 18°C for 24 h.RT-qPCR experiments also found that 10°C cold treatment for 1h,13°C cold treatment for 4h,18°C cold treatment for 24 h,TIAL1,G3BP1 mRNA levels were significantly different(p < 0.001).The results of RT-qPCR experiments and cellular immunofluorescence experiments were consistent.As the stress temperatures decreases,the rate of formation of stress granules also accelerates.2)The formation of stress granules in ZF4 cells is manifested by an increase in the number of stress granules and an increase in diameter.3)After stress treatment at 13°C for 4 h,stress will dissolve rapidly in a short time(about ten minutes)when stress recovery(28°C).The rapid depolymerization process of this stress granule after stress disappearance is also similar to mammalian cells.4)The depolymerization of stress granules is mainly manifested by the rapid dissolution and dissipation of stress granules.Under cold stress,stress granule formation was confirmed in ZF4 cells.We transfected ld4 into ZF4 cells to detect the formation of stress granules in the case of successful expression of ld4.The results are as follows:1)The reverse transcription PCR assay and western blot assay demonstrated that the ld4 gene was successfully expressed in both ZF4 cells at both transcriptional and protein levels.2)Under cold stress,the successful expression of ld4 gene can significantly improve the survival rate of ZF4 cells.3)Immunofluorescence and RT-qPCR experiments of ZF4 cells showed that the successful expression of ld4 under cold stress can reduce the production of stress granules in cells.4)Under cold stress,the expression of p-eIF2 a and p53 in ZF4 cells transfected with ld4 recombinant plasmid decreased.The above results indicate that ZF4 cells can form stress granules under cold stress,and the stress-disappearing stress granules will also depolymerize rapidly.As the temperature decreases,the rate of formation of stress granules also accelerates.This paper firstly found that fish cells form stress granules at cold stress,and the main components are similar to mammalian cells,indicating that stress granules are the general response mechanism of animal cells to cold stimulation.Under cold stress,it was found that LD4 can decrease the levels of tial1 and g3bp1 by inhibiting the phosphorylation of the translation initiation factor eIF2 a,resulting in the formation of stress granules.(stress granules act as a protective mechanism for cells,so the reduction of stress granules will have any adverse effects on cells,so we examined the expression of p53 associated with apoptosis and found that low temperature is in zebrafish cells.The expression of p53 was significantly reduced,indicating that the reduction of stress granules did not affect cell viability.)We suspect that LD4 may play a compensatory role for the stress granules.In this experiment,the preliminary study on ld4 and stress granules at cold stress is expected to provide theoretical support for the study of stress granules in ZF4 cells at low temperature.It is also hoped that the ld4 gene can be used as a candidate for fish cold-tolerant breeding.