Construction Of Puntigrus Tetrazona Eomesa Mutant Using CRISPR/Cas9 Technology

Puntigrus tetrazona is a small tropical cyprinidae fish.In order to study its embryonic development process,genetic breeding,and evolutionary status,The feeding,breeding,early embryo development,chromosome number,DNA content and phylogeny of the P.tetrazona were studied.The research results are as follows:Acidic old water with temperature of 20-30°C and pH=6.5 is its suitable living environment;The best conditions for breeding are:1:1 culture water and distilled water,one female and one male,water temperature 28-29°C,dark treatment 12-15h,Is the best breeding condition with six months to reach sexual maturity.The number of somatic chromosomes is 2n=50;The DNA content of chicken red blood cells is 2.30pg/2c,and its DNA content is 1.50pg/2c.The P.tetrazona mitochondrial genome phylogenetic tree shows that the evolutionary history of the subfamily fish belongs to an earlier fish species.Under the condition of water temperature of 28°C,the whole process of embryo development was observed and photographed by microscope.Like the salmonids,they have experienced the blastoderm,cleavage,blastocyst,primordial,neuroblast,organogenesis and membrane stages.Compared with the model organism zebrafish,the pigment did not form when the P.tetrazona was out of the film for 24hours,and the zebrafish began to form pigments when the film was released at 48 hours,which was not convenient for the observation of the early development.Based on this advantage,it can be a biological model for studying early organ development.It has been reported that the mutation of the T-box transcription factor eomesa leads to the loss of dorsal fin in zebrafish;Mutation of potassium channel protein kcnk5b gene promotes allometric growth of zebra fins;The shh enhancer ZRS on the fifth intron of lmbr1,which knocks out large fragments,causes effects on the pectoral fins or dorsal fins of Oryzias latipes;chordinA is an important gene in the BMP（bone morphogenetic protein）signaling pathway of goldfish（Carassius auratus）,witch mutation causes a double tail fin phenotype.These studies provide a theoretical basis for the amelioration of the P.tetrazona fins.This study cloned the P.tetrazona eomesa gene,ZRS enhancer and the CDS of kcnk5b and chordin.The results are as follows:The coding region of kcnk5b gene,full length 1347 bp,encoding 448 aa amino acid length;coding region of chordin gene,full length 2826 bp,encoding amino acid length 941 aa;eomesa gene containing six exons and five introns,full length 4413 Bp,coding region length 2004 bp,encoding amino acid length 667 aa;ZRS enhancer,full length 4756 bp.The kcnk5b,chordin CDS and eomesa genes were 81%,84%,88%similar to zebrafish,and the amino acid similarities were 79%,87%,and 95%.The nucleic acid coding region of the Sinocyclocheilus grahami gene was 91%,91%,92%,and the amino acid similarity was 88%,94%,95%.These data provide a basis for the study of the molecular mechanisms involved in the development of the P.tetrazona fin.In order to construct a"egg"P.tetrazona without dorsal fin.The CRISPR/Cas9technology was used to knock out the eomesa gene of P.tetrazona,and the efficiency was detected by T7E1 digestion method and TA clone sequencing method.The results showed that the knockout efficiency was between 48%and 64%.At present,65 eomesa F₀ chimeras and 11 F₁ heterozygotes have been obtained.No abnormal phenotype of dorsal fin development has been found.It is necessary to wait for F₂ homozygotes to observe and analyze phenotypes.P.tetrazona is an ornamental fish with good breeding prospects.Variety improvement by biotechnology means,Compared with traditional breeding,it not only saves a lot of time,but also can accurately modify the traits.This study not only accumulated some basic materials in the P.tetrazona breeding,embryo development,chromosome ploidy and genome size,but also explored the P.tetrazona genome editing and breeding.This study provides new ideas for the genetic improvement of ornamental fish.