Phylogenetic Analysis Of KCTD Gene Family And Kctd Mutants Construction In Zebrafish

In recent years,transcriptional activator-like effector nuclease(TALEN),zinc finger endonuclease(ZFN)and,clustered ordered interspaced short palindromic repeats/CRISPR-associated proteins(CRISPR/Cas9)gene editing technologies have been rapidly developed for the editing of various genomes.Among them,CRISPR-Cas9 technology has been widely used with simple and highly efficient features.The construction of animal disease models using the CRISPR/Cas9 system also plays a key role in studying the pathogenesis of human diseases and drug screening.The potassium channel tetramerization domain(KCTD)protein gene family is a conserved gene family.The common feature of this family is that it has one BTB(Bric-a-brack,Tram-track,Broad complex)conservative domain at the N-terminus and a variable C-terminus.The KCTD gene family is widely distributed from invertebrates to vertebrates,with only 5 members in invertebrate nematodes,8 members in fruit fly,and 24 members in vertebrate mice and humans.The biological processes involved in KCTD include transcriptional repression,cytoskeletal regulation and so on,and their proteins play important roles in proliferation,differentiation,apoptosis,and metabolism.Mutations or abnormal regulation of the KCTD genes are associated with various human diseases.Lampreys,the most primitive vertebrates,serve as a bridge over invertebrates and vertebrates in the study on animal evolution.Similar to the amphibians,there is also a metamorphosis process divided into seven stages(M1-M7)in the life history of lampreys.At present,the genomes of Petomyzon marinus and Lethionon japonicum,have been analyzed.This information is important for the study of the origin and evolution of genes and gene families.In this study,we identified 13 and 14 kctd gene members in sea lamprey and Japanese lamprey genomic resources,respectively.The analysis of kctd gene structure in sea lamprey showed that the gene length and exon number are varied significantly between different genes.Four motifs were significantly conserved in all Kctd proteins.Most members of kctd genes are ubiquitously expressed and highly expressed at embryonic development stages.In addition,we also identified the kctd genes in other 12 species representatives from invertebrates and vertebrates and constructed a phylogenetic tree of kctd gene family.The kctd gene family was classified into ten subfamilies based on the clusters in the phylogenetic tree.The results demonstrated that kctd gene family is existed conservatively from invertebrates to vertebrates.Nematode and Fruitfly have 5 and 8 members,respectively.With species evolution from low to high,the number of kctd gene family was increased.The vertebrates possess about 24 members since the appearance of reptiles.The number of kctd gene family was affected in fish as the teleost-specific whole genome 2 duplication event occurs.This study not only enriched the kctd gene family information in lampreys and explored the phylogenetic relationship between different kctd members,but also provided a reference to the functional study of kctd genes in the future.CRISPR/Cas9 technology was used to obtain the knockout mutants of four kctd genes including kctd3,kctd7,kctd8,and kctd14 of the zebrafish,In order that a preliminary study of the function of the kctd genes can be performed on homozygous mutants obtained.We performed statistics on the mutation efficiency using the CRISPR/Cas9 technique,and the mutations efficiencies of the four genes were varied from30% to 90%.Proportion of homozygous mutants in F2 fits to Mendelian' Law of inheritance.Finally,we obtained two types of kctd3 homozygous mutants including-11 bp and-20 bp,two types of kctd7 homozygous mutants including +19 bp and-19 bp,one type of kctd8 homozygous mutant+13 bp,and one type of kctd14 homozygous mutant-8 bp.Using semi-quantitative RT-PCR with ?-actin as an internal standard,kctd gene expression were investigated for different tissues(gill,skin,eye,ovary/testis,heart,muscle,liver,kidney,spleen,and brain)and different stages(day 1,2,3,5,10,20,30,45,and 60)of zebrafish..Kctd3 and kctd7 all showed high expression in various tissues,kctd8 and kctd14 expression showed significant tissue specificity,kctd8 expression was high in eyes,testis,and brain,and kctd14 was highly expressed in ovary,muscle,and kidney.The expression patterns of kctd3,kctd7,kctd8,and kctd14 were similar in zebrafish at different developmental stages,showing that on the third day the expression was significantly higher than on the first and second day,From day 5 to day 45,the expression level was significantly higher than those on day 1 and 2,but after 45 day,the expression level showed a downward trend and specificity.These results are of significance for the further analysis of function of the kctd genes and understanding of the pathogenesis of some human diseases caused by kctd mutation,and for the prevention,diagnosis and treatment of human diseases.