Research On Characterization And Preliminary Function Of Suppressor Of Cytokine Signaling 1 In Megalobrama Amblycephala

Blunt snout bream(Megalobrama amblycephala)is known as an important representative of the freshwater aquaculture economicspecies(Cypriniformes,Bo subfamily)in our country.Due to good growth characteristics(wide range of food,low breeding costs,high survival rate),it is become more and more popular.As a principal herbivorous species in freshwater fish polyculture systems,blunt snout bream(Megalobrama amblycephala)is widely favored as a delicacy in China,whose output is more than 800,000 tons in 2017(FBMA.,2018).it has a big size with a long cycle of sexual maturity.Traditional way of breeding needs higher cost and longer time.The genome of Megalobrama amblycephala still exists quite a number of main effect genes which function is unknown.Study their function has important theoretical significance and application value for economic properties such as reproduction,growth,development and tolerance of Blunt snout bream.The suppressor of cytokine signaling 1(SOCS1)is an essential feedback regulator extensive involved in many different cytokine signaling pathways,regulating immune and growth of organism.To date,the study of complement C3 in mollusk focused on sequence and structure analysis.To date,the study of SOCS1 in teleost focused on sequence and structure analysis,comarped with Mammal,Functional studies are not in-depth.In this paper,SOCS1 s had been obtained in Megalobrama amblycephala and analysed on gene structure,mRNA expression,and functional exploration:1.we identified and characterized the full-length closely related but distinct SOCS1 genes(SOCS1a and-1b)in blunt snout bream(Megalobrama amblycephala).The SOCS1 a was 1065 bp in length,encoding 201 aa.The overall SOCS1 b was 817 bp in length,encoding 197 aa.The bioinformatic analysis results showed that duplicated socs1 shared majority conserved motifs with other vertebrates.The socs1 a and –1b mRNA were abundantly expressed in most of the blunt snout bream tissues.The socs1 a mRNA were strongly expressed in the heart,eye,kidney and spleen,but were found to be relatively low in the intestine and liver.On the other hand,the expression of socs1 b mRNA was significantly high in the muscle and spleen and relatively low in the intestinal tract,liver,skin and heart.Additionally,duplicated socs1 mRNAs were detected in all stages of embryogenesis and expressed in a ubiquitous pattern.Whole-mount in situ hybridization demonstrated that socs1 a mRNAs were detected in the brain at 12 hpf and 24 hpf,and in the notochord and eyes at 36 hpf,while socs1 b mRNAs were detected in the brain at 12 hpf and detected in the gut at 24 hpf and 36 hpf.The results of hGH treatment experiment showed that SOCS1 a and 1b mRNAs were upregulated markedly in the kidney,muscle and liver.2.The obtained genes of blunt snout bream,SOCS1 a and SOCS1 b,were used as genetic editing objects.Then we screened for appropriate target synthetic gRNA(guide RNA)by online analysis,It contains the main site sequence,which is responsible for identifying the target gene/target sequence;Cas9 is responsible for cutting the target,causing DNA double strand breakage(DSB).According to the mixture injection(gRNA and Cas9 protein)in the 1-2 cell stage embryos.efficiency targets is SOCS1a-target 2 with enzyme digestion efficiency at about 35%.SOCS1 s higher efficiency targets is SOCS1b-target 2 with enzyme digestion efficiency is 20%.By detecting the effectiveness of the target,it was found that the bases of the SOCS1 a and SOCS1 b genes were deleted and misplaced,indicating that the CRISPR/Cas9 system is effective for the knockout of the SOCS1 gene.After the detection of 3-month-old F0 generation blunt snout bream,the F0 generation individuals with the mutation of the target sequence were successfully screened.Compared with the wild type(WT),the overall growth of heterozygosis mount was largely retared abnormal inflammatory.There are significant variations in the expression levels of the typical inflammatory cytokines,such as IL-6 and TNF-?,between the wild-type control and heterozygous duplicated socsl-deficient mounts,rather than IL-1?.After knock-out SOCS1 a and SOCS1 b blunt snout bream were challenged by intraperitoneal injection with A.hydrophila,In contrast to the wild type,significant increases in levels of IL-6 and TNF-?mRNA were observed in both SOCS1 a and SOCS1 b heterozygous mutants.The dupliacated socs1 knockout blunt snout bream have been successfully obtained by the CRISPR/Cas9 gene editing system,which provides a basis for further study of the socs1 gene.Meanwhile,our experimental results provide a basis and reference for the study of CRISPR/Cas9 gene editing techniques in other aquaculture species.