Study On Goldfish Tgf2 Transposon Transposition Efficiency And Hypoxia-Induced Expression Analysis Of HIF3α Gene In Megalobrama Amblycephala

Transposon are DNA fragments that can freely jump(replicate or shift)on the genome,and the process of movement is called transposition.According to the transposition mechanism,scientists have divided transposons into two categories: class I is a retrotransposon and class II is a DNA transposon.The insertion mutagenesis strategy of transposons has important research prospects in the selection,function interpretation and transgene of major genes in fish.The main object of this experiment is the Tgf2 transposon,which is a DNA transposable element with autonomous transposable activity found in our laboratory.This study focuses on improving the nuclear efficiency of the transposase protein and then increases the Tgf2 transposition efficiency.The purpose is to artificially interfere with the transposable element to enhance the transposition function,establish a theoretical basis for the integration and stable expression of foreign genes,and provide support for insertion of mutagenesis in fishes.We constructed pEGFP-Tgf2 TP transposase expression vector and pEGFP-TP-SV40 NLS transposase plasmid containing the nuclear localization signal of SV40 large T antigen,to study whether the the nuclear and transposition efficiency could be increased by artificial interference transposition element.The Tgf2 transposon donor plasmid pTgf2-EF1α-eGFP with the green fluorescent protein was co-injected with the mRNA of pEGFP-Tgf2 TP and pEGFP-TP-SV40-NLS plasmid into the zebrafish fertilized egg.After in vitro culture,the fluorescence microscope was used to observe the GFP reporter at different stages of embryonic development,and the positive rate was detected by sequencing analysis.Hypoxia-inducible factors play key role in resistance to hypoxia,and their activity is regulated by the α-subunit.HIFs can activate a series of low-oxygen-sensitive genes for their transcription in the downstream to regulate a variety of physiological processes,including the energy metabolism,erythropoiesis,angiogenesis,cell proliferation and apoptosis,to keep the body maintain homeostasis under hypoxic conditions.At present,the researches on HIF1α and HIF2α are more in-depth in human,mammal and fish,but the structure characteristics of HIF3α and its regulation mechanism under hypoxia condition remain limited.We hope that by studing the structure of HIF3α and its hypoxic function,we can explore its regulatory mechanism of intolerance to hypoxia,and then provide a theoretical basis for cultivation hypoxia-resistant strain of blunt snout bream.To this end,we constructed an expression vector pCS2-HIF3α-EGFP containing blunt snout bream HIF3α to study the nuclear localization under normoxic and hypoxic conditions,which may provide a theoretical basis for the study of hypoxia tolerance of blunt snout bream.The purpose of this study was to detect the expression levels of HIF3α at the transcriptional level and protein level in order to find the mechanism of hypoxic regulation and provide a theoretical basis for the study of the resistance to hypoxia.Our findings are as follows:(1)The results showed that the positive rate of plasmid containing SV40 NLS was 12.7% higher than that of the control group,indicating that SV40 NLS can help improve the integration efficiency and the transposition efficiency of Tgf2 transposable elements.When transfected into HeLa cells,the green fluorescence of pEGFP-Tgf2TP-SV40 NLS transposase were more concentrated in the nucleus.Compared with the control cells transiently transfected with the pEGFP-Tgf2 TP transposase plasmid,the efficiency of nuclear translocation is 51.63%,which is 11.58% higher than that of the control group.It is suggested that SV40 NLS can help to improve the nuclear efficiency.The positive rate of zebrafish microinjected with the pEGFP-TP-SV40 NLS transposase plasmid was 60.8%,compared with 48.1% in the control group,the positive rate increased by 12.7%,indicating that SV40 NLS could help to improve the integration efficiency.(2)The blunt snout bream HIF3α belongs to the bHLH family and has PAS,PAB,PAC,ODD,and TAD domains that are unique to this family.After transfection with HeLa cells,we found HIF3α protein concentrated in the nuclei under normoxic and hypoxic condition.Hypoxia treatment resulted in a significant increase in the expression of HIF3α at protein levels in the test tissue.In addition,there was a nuclear localization signal sequence at the C-terminus of the blunt snout bream HIF3α protein,which may help HIF3α protein enter into the nucleus under hypoxia conditions.