| As a heterothermic animal,temperature is one of the most important environmental factors in the survival process of fish.Which not only determines the distribution of fish,but also directly and indirectly affects the physiological function and behavior of fish.At the cellular and molecular level,low temperature can not only greatly reduce the rate of enzymatic reaction and transport in the body,but also induce protein denaturation,inhibit transcription and translation,destroy cytoskeleton structure and change the permeability of cell membrane.In addition,cold stress also changes the characteristics of cell lipid bilayer,including phase transformation and fatty acid composition.The gill tissue of fish is directly exposed to the external environment,which is the first organ of fish to perceive the external environmental pressure.Gill plays an important role in adapting fish to the changes of external environment temperature and regulating physiological responses in vivo.CRISPR/CAS9 technique was used to knock out bmp10（bone morphogenetic protein 10,bmp10）and dusp1（dual-specificity phosphatase 1,DUSP1）genes in zebrafish early in the experiment.It was found that bmp10 and dusp1 were controlled to regulate the apoptosis of gill tissue in zebrafish under low temperature,but the molecular mechanism of apoptosis was still not clear.Bmp10 is a member of the TGF-βsuperfamily.Previous studies have demonstrated that bmp10 plays an important role in the development of the mouse heart,but there is still no report in the study of zebrafish.In our laboratory,the CRISPR/CAS9 technique was used to knock out the bmp10 gene of zebrafish and the F2 generation that can stably inherit was successfully obtained.It was found that bmp10 played an important regulatory role in the apoptosis of fish gill tissue cells at low temperature.However,its specific molecular regulation mechanism is still unclear.Therefore,the bmp10-/-homozygous mutant zebrafish obtained in the previous study was the subject of the study and found that compared with the wild type zebrafish,the expression level of P53protein in the bmp10-/-homozygous mutant zebrafish was significantly lower than that in the wild type zebrafish.increase.At the same time,bmp10-/-homozygous mutant zebrafish mRNA expression level of P53 and its target genes will increase rapidly at low temperature environment,and the apoptotic tissue of axillary tissues will cause serious damage,accelerate the bmp10-/-homozygous mutant zebrafish death.Simultaneously,the wild-type zebrafish myelinated by myelin7-gfp fluorescence cardiomyocytes was hybridized with the bmp10+/-heterozygous mutant zebrafish,and finally a bmp10-/-homozygous mutant zebrafish specific for green fluorescence-labeled cardiomyocytes was obtained.Inverted fluorescence microscopy revealed that the bmp10-/-homozygous mutant zebrafish had reduced cardiomyocytes and abnormal cardiac development,eventually accelerating the death of the bmp10-/-homozygous mutant zebrafish,further demonstrating bmp10 in the zebrafish heart also plays an important regulatory role during development.Dual-specificity phosphatase-1（DUSP1/MKP1）is a member of the threonine-tyrosine dual-specificity phosphatase family.which was originally found in cultured murine cells.Previous studies have shown that dusp1 functions by mediating the dephosphorylation of extracellular signal-regulated protein kinases.And in the cell proliferation,differentiation and apoptosis process has an important regulatory role,the previous study found that the dusp1-/-homozygous mutant zebrafish apoptosis in the low temperature environment significantly increased the cell apoptosis,However,the relevant molecular regulatory mechanisms that cause apoptosis in sputum tissue are not yet clear.In this research,we examined the level of the previously duspl-/-homozygous mutant zebrafish protein and found that the duspl-/-homozygous mutant zebrafish caused a significant increase in apoptosis of the iliac crest cells at low temperatures,mainly due to MAPKs.Phosphorylation of P38 and ERK in the pathway increases,which accelerates the apoptosis of the nevus tissue and causes damage to the necrotic tissue.Gsdf is a specific member of the TGF-βsuperfamily,which is mainly expressed in male gonads of teleosts.However,the specific molecular regulation mechanism in testis development is not clear.Therefore,in this research,a Tg（Crystal-pro-gsdf-2A-egfp）luciferase reporter plasmid started with the mouse gamma-crystalin promoter was constructed using a microinjection technique to inject Tg（Crystal-pro-gsdf-2A-egfp）plasmid to 1-cell stage in zebrafish fertilized embryos.Observed and screened under inverted fluorescence microscope,finally obtained 19 chimeric F0 generations capable of specifically expressing green fluorescent protein（GFP）in the zebrafish eye lens,and then the zebrafish expressing the green fluorescent protein was wild-type（WT）non-transgenic zebrafish were crossed and eventually F2 generations that could stably inherit and express green fluorescent protein were obtained.In summary,This research mainly revealed the molecular mechanisms involved in the regulation of apoptosis in tissues of zebrafish bmp10 and dusp1 during cold stress,which provided an important theoretical basis for studying the effects of low temperature on fish growth and development.At the same time,a transgenic Tg（Crystal-pro-gsdf-2A-egfp）zebrafish strain was successfully established,providing a new method for observing and studying the role of gsdf in the testis development process and molecular regulation mechanisms.In conclusion,the research of the effects of related genes in low-temperature environments on the normal growth and development of zebrafish and the functional roles of sex-key genes in zebrafish male and female differentiation are important for understanding fish adaptation to cold evolution and fish sex differentiation.at the same time,it also plays an important biological foundation for the cultivation of cold-resistant,anti-freezing,and full-fledged economic fish. |
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