Screening And Gene Analysis Of Zebrafish Induced By Plasma Mutagenesis (ARTP) At Room Temperature And Pressure

Fish farming accounts for a large proportion in China’s aquaculture.In order to obtain individuals with good traits,fish mutagenesis breeding technology has gradually attracted the attention of the majority of scholars.Mutation breeding generally uses physical or chemical methods to cause fish DNA damage,so as to produce heritable variation and obtain excellent traits in a relatively short period of time.However,the traditional mutagenesis method has the disadvantages of poor safety and low frequency,so it is very necessary to find a safe and efficient mutagenesis method.In this paper,zebrafish was taken as the main research object,and wild-type zebrafish was taken as the experimental parent to conduct in vitro artificial insemination operation and mutagenesis treatment for the obtained zygotes.The main treatment methods were as follows:ARTP treatment was performed on the zygotes when they developed to cell stageⅠ,and the treatment time was set as:90,180,270,360,450,540,630 and 720 s.Then the fertilization rate,white egg rate,hatching rate,malformation rate and other relevant indicators of the treated fertilized eggs were counted.The F₁generation was bred with the wild type after 3 months,and the F₂generation produced was screened for traits,and the whole genome of the F₂generation with special traits was resequenced,and the mutant genes were analyzed accordingly.（1）After processing zebrafish embryos with ARTP,the fertilization rate,white egg rate,hatching rate and malformation rate were counted.It was found that ARTP mainly had a great influence on the fertilization rate of zebrafish embryos.After the statistics of experimental results,it was found that the fertilization rate of wild blank control group could reach 85%～100%.The fertilization rate of different treatment groups was lower than that of wild blank control group,and the fertilization rate was about 38%～82%.The experimental results indicated that ARTP had a certain blocking effect on the fertilization process of zebrafish embryos.The white egg rate of zebrafish embryos treated with ARTP for a long time can be reduced to a certain extent.The white egg rate of the blank control group is about 4%～11%,and that of the experimental group is about 6%～32%,which proves that ARTP can indeed affect the normal development of zebrafish embryos,resulting in the development stop or even death of zebrafish embryos within 8～24 h of development.And that eventually leads to white eggs.In addition to the above results,ARTP mutagenesis may also cause abnormal development of zebrafish embryos,and in serious cases,the hatching rate of zebrafish embryos will decrease and the malformation rate will increase.（2）in order to further screening mutant zebrafish,after dealing with the mutation of embryos for artificial incubation,waiting for the zebrafish embryo to 3～5 months hybridization experiments,the results found that exist in the 720 s treatment group sperm activity of mutant zebrafish,wild type zebrafish males mate after fertilization rate is about 87%～99%,However,the fertilization rate of mutant zebrafish after normal mating was less than 10%,and the results were different（P≤0.001）.（3）The fertilized eggs of the experimental group were conventionally cultured,and the hybridization experiments were carried out after their development to sexual maturity,and the offspring were screened.After a large number of hybridization experiments,two mutant traits were obtained,namely cardiac cavity malformation and mandibular dysplasia.Heart cavity malformation of zebrafish larvae in a stered microscope can be clearly observed its heart cavity parts),in order to further determine the mutant zebrafish heart cavity abnormal degree,using histology slice staining,after the analysis of the results found that the mutant zebrafish ventricular atrial differentiation is not obvious,and intentional cavity effusion.The larvae of normal wild-type zebrafish inflate their swim bladder at 6 DPF after fertilization and begin to swim flat for feeding.However,even at7 DPF after fertilization,the swim bladder of zebrafish larvae with mandibular mutant is still unable to inflate,swim flat and feed,and die with time.Under stereomicroscopy,it was found that the anterior part of the lower jaw was protruding from the head of the normal zebrafish larvae,while the anterior part of the lower jaw was shorter than the head of the zebrafish larvae with abnormal mandible development.In order to further clarify the difference in traits,alcin blue staining was carried out for the mutant zebrafish,and the results showed that the mutant zebrafish lost MCS cartilage in the mandible,the keratoglossia cartilage was invaginated,and the keratoglossia bone could not expand.（4）Whole-genome resequencing of mutant male zebrafish and wild-type male zebrafish with reduced sperm motility after ARTP treatment showed that ARTP could effectively improve the mutation efficiency of the organism.Concrete improve mutation types mainly include the number of single nucleotide sequence mutation in exon,exons give genetic termination codon mutation in number,the number of synonymous mutations in exon,lack in exon protein-coding box change the number of mutations,is located in the exon region number of copy number variation,is located in the areas of introns copy number variation,increased copy number digits,the copy number increases the length,etc.