Transcriptome Analysis Of Notch1a In Innate Immune Response In Zebrafish(Danio Rerio) Larvae Challaged By Vibrio Parahaemolyticus

Zebrafish（Danio rerio）is an important animal model,and has advantage in studying the innate immune response.It has only innate immune responses within 3-4 weeks after fertilization and does not have adaptive immune responses.Notch signaling pathways play an important role in the growth and development of embryos,and previous studies have shown that Notch signaling pathways also play an important role in adaptive immune responses.However,the mechanism of Notch signaling pathway in innate immune response is unclear.Vibrio parahaemolyticus is a main pathogen of human and animal,which can infect both aquatic animals and humans.The consequences are serious,but the mechanism is still to be studied.In this paper,3 days post fertilization（dpf）larvae were infected with Vibrio parahaemolyticus for 2 hours.The differentially expressed genes were analyzed by transcriptome sequencing.The mechanism of Notch signaling pathway was studied preliminarily.Firstly,by comparing the transcriptome data of the two groups with immersion infection and tail vein injection infection,there are 602 differentially expressed genes are found in immersion group,427 genes are down-regulated,and 175 genes are up-regulated,in which there are 15 immunity related genes including il11 a,ccl34,tnfb,ccl20 a,cxcr4a,cxcl3,fosl1 a,cmya5,c3 a,c8a,c8 b,mmp13,ctslb,atf3 and arg2;359 differentially expressed genes in injection group,246 genes are down-regulated,and 113 genes are up-regulated,in which there are 20 immunity related genes including il1 b,il6,il34,tnfa,ccl19 b,cxcr3,cxcl3,cxcl18 b,tnfb,ccl20 a,ccl35.1,irak3,nfkbiaa,fosl1 a,fosab,rel,c5,ncf1,ctslb and mpeg1.Genes related to cytokine contained il11 a and ccl34 which are significantly down-regulated,and tnfb,ccl20 a,cxcr4a and cxcl3 are significantly up-regulated.In immersion group,il1 b,il6,il34,tnfa,ccl19 b,cxcr3,cxcl3 and cxcl18 b are significantly down-regulated,tnfb,ccl20 a and ccl35.1 are up-regulated in injection group.Genes related to complement system contained cmya5,c3 a,c8a and c8 b are significantly down-regulated in immersion group,and the c5 gene is down-regulated in injection group.The 602 DEGs in immersion group are enriched in 632 GO terms and 101 KEGG pathways.The 359 DEGs in the injection infection are enriched in 106 GO terms and 56 KEGG pathways.It is indicated that the differentially expressed genes and pathways are different in two group.The immersion infection can activate complement system and can cause a systemic immune response process,and the GO terms and KEGG pathways are more extensive.The injection infection can activate the TLR pathway firstly,and then genes related to cytokines are widely expressed,affecting the GO terms and KEGG pathways.We use CRISPR/Cas9 system to knock out notch1 a.Then we use Vibrio parahaemolyticus to infect 3 dpf larvae of notch1a^-/-2h.986 genes are differentially expressed in the transcriptome data.The expression of transcription factors irak3,nfkbiaa and other genes in the TLR signaling pathway are significantly up-regulated.Comparing with the data of the wide type zebrafish group infected by Vibrio parahaemolyticus,the downstream genes of TLR signaling pathway,cnksr3,appa,anxa1 c,pcdh11,neo1 b,arr3b,tiaf,cxcl3,il1 b,il34 and ccl35 are also significantly up-regulated.Genes of il34 and ccl35 are significantly up-regulated in both groups,but there is also a significant difference between the two groups.indicating that the immune-related genes arr3 b,tiaf,cxcl3 and il1 b may not be directly regulated by Notch1 a,il34 and ccl35 are regulated by the Notch1 a signaling pathway.The expression of the genes,cxcl18 b,cxcr3,irak3,nfkbiaa,cfb,myhb,myh11 a,serpinf2a,ctss2 and ctslb are significantly down-regulated in wild type group and up-regulated in notch1a^-/-group.There is no significant difference in immune-related GO term in notch1a^-/-group,whereas in the wild type group the GO enrichment is significantly difference.We analysis the KEGG pathways and there is no difference of immune-related pathways in both groups,indicating that infection for 2h don’t have a significant effect on the body,at the beginning of the innate immune response,and Notch1 a may inhibit the activation of the TLR pathway by inhibiting the irak family,and play a negative role in the regulation of the innate immune response when 3 dpf zebrafish larvae are infected by Vibrio parahaemolyticus for 2h.In one word,we have obtained different genes of zebrafish in immersion infection and tail vein injection.We also find that the Notch1 a signaling pathway plays an important role in innate immune response.Our results provide new evidence and research ideas for the research of Notch signal pathway in innate immune response.