Establishment Of Zebrafish Kbtbd12 Knockout Model And Preliminary Analysis Of Related Biological Functions

The KLHL protein superfamily plays an important role in muscle development and disease occurrence.Several members of the family,such as KLHL31 and KBTBD13,act as substrate-specific adapters for ubiquitination,regulating protein stability and participating in sarcoma formation.But until now,the functions of other family members are unclear in muscle development and disease occurrence.According to the gene expression data of normal tissues,it was found that KBTBD12 gene was highly expressed in the heart and muscle tissues of vertebrates,such as human,mouse and zebrafish.We speculate that it may be related to the development of heart and muscle and the occurrence of related diseases.Further analysis showed that KBTBD12,a member of the KLHL protein superfamily,is highly conserved during the process of biological evolution.In order to investigate the function of the gene in the vertebrate heart and muscle tissue,we used the zebrafish,an ideal animal model,for further studies.The zebrafish kbtbd12 gene is located in zebrafish chromosome 6 and contains 6 exons.The results of in situ hybridization showed that the gene was highly expressed in muscle tissue and weakly expressed in heart during embryonic development of zebrafish.The CRISPR/Cas9 gene editing technology was used to design and synthesize the corresponding sg RNA at each target site of the upstream and downstream exon 1 of the gene,and then microinjection embryos were used for target shooting.Through the injection of progeny for genetic detection,and F₁generation mutant screening,we successfully screened the zebrafish knockout strain with 74bp deletion of exon 1,namely CRISPR-kbtbd12(-74 bp).Western blot analysis showed that the mutant homozygote was deficient in kbtbd12 protein.These results indicated that the zebrafish kbtbd12 gene knockout model was successfully established.Phenotypic and functional analysis of the kbtbd12(-74 bp)mutant homozygous showed that,compared with the wild-type control group,there were different degrees of slow muscle development in the kbtbd12(-74 bp)mutant homozygous,including the mutant phenotype of myosegmental development.In situ hybridization analysis showed that the expression of skeletal muscle development marker gene myod1 was down-regulated in mutant homozygous zebrafish embryos compared with wild-type zebrafish embryos.Western blot analysis of protein expression showed that compared with wild-type zebrafish embryos,the expression levels of ubiquitination response related substrate proteins ckap4 and mybpc2 were up-regulated in mutant homozygous zygotes,indicating that the abnormal muscle development of zebrafish caused by kbtbd12 gene knockout may be related to ubiquitination.The above findings will be important for exploring both the function and mechanism of action of kbtbd12during zebrafish muscle development.