Discovery Of Genes Related To The Ossification Of Intermuscular Bone In Yellow River Carp

Carp is an indispensable freshwater cultured species and is farmed in many countries.China is the most farmed among them,accounting for 72%of the total.The Yellow River Carp is a naturally formed population in the Yellow River basin,and its golden scales red tail body slender taste delicious,is one of China’s four freshwater fish.However,most of the cyprinids represented by Yellow River carp are rich in intermuscular bone（IB）,which are spicule-like bones exist in the myosepta on both sides of the vertebrae.It is dense and difficult to remove,which not only brings certain safety risks to eat directly,greatly reduces its consumption value,but impedes processing of machining,which highly hindered the development of freshwater aquacultureBy optimizing the Yellow River carp IB staining method,the five key nodes（not appear IB,first appearance of IB in the tail,the number of IBs increased,first appearance of IB in the head,and completely ossification of IB）in the process of IB ossification of Yellow River carp were identified.High-throughput sequencing was performed on the tail tissue transcriptome,and unigene were compared with the carp genome,annotated by GO enrichment analysis and KEGG enrichment analysis.After the expression profile clustering,relevant genes involved in IB ossification of Yellow River carp were screened,and expression profiles of 16genes related to bone development were verified by q RT-PCR.Edit bmp3 by CRISPR/Cas9 to obtain mutant chimera zebrafish to verify the function of the gene,the main research results are as follows:（1）The alizarin red staining method was optimized from dyeing time,temperature,concentration of H₂O₂,fading time and glycerol transparency time,and achieved good results in IB staining of Yellow River carp.This method can quickly stain the IB of carp,which is conducive to the study of IB morphology.（2）Five key nodes of IB development of Yellow River carp were identified by staining of IB larvae.S1:not appear IB;S2:first appearance of IB in the tail;S3:increased number of IBs;S4:first appearance of IB in the head;S5:complete development of IBs.This study selected the tail tissue of the five key stages of IB as the research object;The transcriptome analysis showed that a total of 781,798,980 clean reads,67,513unigene were obtained.The sequencing quality of 97.78%of the sequences was above Q20,GC content was47.45%,the number of GO functional annotated genes was 32,767,and 6,499 genes were enriched in KEGG signaling pathway.（3）Pay attention to the dynamic changes and difference comparison of the transcriptome in the process of IB formation,and found that a large number of genes show dynamic changes from the beginning of the ossification of the IB to the complete ossification,which shows that the ossification of the IB is a complicated process.GO Enrichment analysis showed that DNA replication,cell migration regulation,cell migration,Wnt signaling pathway and other cell movement and proliferation-related genes have a vital role in the early stage of IB ossification;sarcomere,myofibril,myofilament and movement-related Genes are closely related to the late development of IB.KEGG analysis results show that genes in the cell cycle and mismatch repair pathways are likely to be related to the early differentiation of IB;Fox O,VEGF and ECM pathways are enriched in the late IB development,suggesting that these genes are Participate in IB ossification.（4）The KEGG metabolic pathway enriched genes are de-redundant,the differentially expressed genes are divided into 6 different profiles according to the dynamic expression.Each profile was re-enriched on the KEGG pathway,and the results showed that CCI,VEGF,TGF-β,Fox O,MAPK,ECM were screened in profiles 2 and 4,and the 6 pathways related to bone development were significantly enriched on profile 2and profile 4.The trend of profile1,profile2 and profile4 is analogous.The results illustrate that CCI,VEGF,TGF-β,Fox O,MAPK,and ECM pathways may be involved in the regulation of IB ossification,but the specific mechanism needs to be further study.（5）The study screened 295 differential genes according to the expression profiles of profiles 1,2 and4 and 6 bone development-related pathways;according to|log2FC|>0,S1Vs S2＿padj<0.05 or S1Vs S3＿padj<0.05,and FPKMmax<10 screened out 194 possible candidate genes for IB ossification.（6）On the basis of the CRISPR/Cas9 system,targets are chosen and primers are designed,and the technology finally constructs a zebrafish chimera of bmp3 mutant.In this experiment,it was observed that the edited zebrafish post of the hatching was late,and some individuals showed obvious spine and tail curvature,but it has not been determined that it is caused by the loss of bmp3.From the perspective of tissue staining,comparative transcriptomics and CRISPR/Cas9,this study explored the changes in the expression levels of key nodes in the IB of Yellow River carp,and found that multiple pathways which related to bone development may play an indispensable role in the ossification of IB.A number of candidate genes involved in IB ossification were mined,and bmp3 mutant zebrafish was constructed with CRISPR/Cas9 technology.