Effects Exogenous P204on Zebrafish’s Innate Immune And Construction Of P204Transgenic Vectors

There are two kinds of immune systems involved in host defense against pathogen infection, including innate immunity and adaptive immunity. The innate immunity is the first line of defense and its key process is the recognition of the conserved pathogen-associated molecular patterns (PAMPS) in pathogens by pattern recognition receptors (PRRs). Toll-like receptors (TLRs) as important extracellular and intracellular receptors, participate in the activation of NF-κB and IFN-β signaling pathway which lead to the inflammation and host antiviral responses. Innate immunity and adaptive immunity are not two independent systems. On the contrary, the crucial molecules and signal path of innate immunity can activate adaptive immunity. Within the mechanism, TLRs signal path is the bond jointing innate immunity to adaptive immunity. Zebrafish as same as humans has both innate immunity and adaptive immunity. Researches on the adaptive immunity system of zebrafish have last for many years and make significant achievement along with the great improvement of research techniques. However, the study on the innate immunity of zebrafish is just starting out. Innate immunity system is the first line for organism to defend microbial pathogens and has important effect on the activation of adaptive immunity. Therefore, not promoting the understanding of innate immunity system of zebrafish, more in-depth studying on the innate immunity of zebrafish can provide study thinking and tools for the immunity research of vertebrates from the view of evolution. Moreover, since zebrafish has many advantages such as small size, easy feeding, short sex maturity cycle, easy reproduction and high rate of reproduction, lucid embryo easily observed, and expedient operation on the embryo, it has been broadly used in the study of functional genes.We used the model animal zebrafish, and microinjected the exogenous gene p204vector into the zygotes of zebrafish. By observing the expression of the report gene GFP, we found that p204was expressed in the embryo of zebrafish from the stage8hpf especially mainly concentrating in the chest and stomach. Subsequently, p204was highly expressed during the stage8hpf-8dpf and began to reduce after8dpf until about lldpf with complete disappearance of fluorescence. The injection of p204-EGFP could induce the change of phenotype of zebrafish, and cause deformity such as bigger abdomen and curly tails and so on.By selecting the injected embryos expressing green fluorescence proteins, quality real time PCR was performed to test the genes tbkl and sting related to the innate immunity. And we discovered that after injecting p204vector the expressions of tbkl and sting is apparently up regulated compared to the controls during the stage8hpf-48hpf, but have no change in the stage4hpf. In addition, we performed RNA in situ hybridization of relevant genes, and the result is identical with the results of real time PCR. In order to gain the zebrafish mutants whose genomes fused with p204, we successfully used the transposon tol2carrying p204to construct the vector tol2-p204and transformed it along with TP transposase into zygotes of zebrafish. Our study results suggest:1)the exogenous gene p204can influence the development of zebrafish and cause the abnormal phenotype to a certain extent;2) p204can affect the innate immunity system of zebrafish and participate in the signal path;3) The vector tol2-p204was constructed successfully and injected into the zygotes of zebrafish. But the expression of p204was unstable.This study provides new proof for the research of innate immunity of zebrafish and related molecular mechanism and the evolutionary relation between zebrafish and vertebrates.