Impact Of CfGnRH Gene Knock Out On Reproductive Capability In Channel Catfish,Ictalurus Punctatus

Targeted gene editing is an efficient application for genomic functional studies.Transcription activator-like effector nucleases(TALENs)have promptly emerged as one of a most vigorous gene editing technology.It can be designed to target nearly any site in any genome with easier operation,increased efficiency and lower cost.TALENs-mediated mutations have been reported in various species of fish,including zebrafish(Danio rerio),medaka(Oryzias latipes)and yellow catfish(Tachysurus fulvidraco).Gene editing has great potential in improving varieties,improving productivity,and treating diseases.Channel catfish(Ictalurus punctatus)is a major freshwater aquaculture production species in the United States.Contrastingly,the catfish industry is being severely challenged during these years as results of higher operating costs,disease and the competition from cheaper imported frozen fish.Many genetically modified animals include fish utilize transgenes to increase yield significantly,however,followed by the widespread concern about the ecological and genetic effects.Sterilization technologies of catfish can be used to prevent the ecological security risk of the leakage of genetic fish.In the early stage of this study,TALEN technology and double electroporation technology were used to knock out cfGnRH gene of germ cell to obtain P1 generation.In this experiment,P1 generation was crossed with wild type channel catfish to obtain F1 generation.P1 generation and F1 generation of channel catfish were used as materials.TALEN plasmids targeting the cfGnRH gene were delivered into fertilized eggs with electroporation technique to knockout cfGnRH gene.In this study,the mutation analyses were compared between 6-month-juveniles and 3-year-old adult fish.Enzyme-linked immunosorbent assay(ELISA)and fish spawning experiments were utilized to detect the presence of sexual hormone and evaluate the reproductive capability of mutant fish,non-mutant fish and wild type fish.The CEL-I mutation assay were utilized to detect the mutation of F1 juveniles.The results paved the foundation for preventing the potential negative effects of hybridization,toxicity and gene modified fish on the ecological environment.The major results were as follows:(1)Statistics of sexual maturation rate.In this research,the sexual maturation rate of channel catfish in the TALEN group was analyzed when they grew up to 3-year-old.The abdomen of sexual mature females in the spawning season,were usually swelling,soft and flexible.The reproductive hole shows redness,slightly outward protruding.Sexually mature males generally have a deep color of body.The reproductive nipples are large and hard.The head muscles are developed.The channel catfish in the experimental group were divided into mutant type and non-mutant type.The fish of mutant type were found mutation targeted cfGnRH gene.In contrast,the fish of non-mutant fish were not found mutation targeted cfGnRH gene.The results showed that in the same circulating system,there were 31.25% of the mutant fish showed sexual maturity characteristics,while there were 83.33% of the non-mutant fish showed sexual maturity characteristics.The sexual maturation rate of mutant females(20%)was little bit lower than mutant males(36%).The sexual maturation rate of non-mutant females(100%)was little bit higher than non-mutant males(75%).This might be due to the negative effect on the gonadal development from cfGnRH gene knockout.Also,gonadal development depends on the regular pulse of hormone.The demands for the hormone pulse of females might be more stringent.(2)Mutation analysis.In this study,the CEL-I assay was utilized to detect mutations in channel catfish when they grew up to 3-year-old.5 mutant fish,6 non-mutant fish and 4 wild type fish were randomly selected.Pelvic fin and barbel were sampled for use with Surveyor Mutation Detection Kit(Integrated DNA Technologies,Coralville,IA)to detect mutations.The mutation detection results were kept consistent with those at 6-month-old.Therefore,with the migration of time,gene mutation situation didn't change,and it could be considered that gene mutation could stably exist.The stable presence of mutations ensures the function of the cfGnRH gene is affected for a long time.Then the development and the maturation of the gonad and the generation of gametes were repressed.(3)Spawning experiments.In the first stage,some sexually matured 3-year-old channel catfish were selected to be paired with sexually matured wild type channel catfish,include 7 mutant males,1 non-mutant male,4 mutant females and 1 non-mutant female(wild type?×mutant type?,wild type?×non-mutant?,mutant?×wild type?,non-mutant?×wild type?).These mutant and non-mutant fish were numbered from 1-13.Only wild type females were injected with LHRHa.In the second stage,mutant and non-mutant females were also injected with LHRHa.In the third stage,2 mutant females were crossed with 1 mutant male and 1 non-mutant male,respectively(mutant?×mutant?,mutant?×non-mutant?).1 non-mutant female was crossed with 1 mutant male(non-mutant?×mutant?).Both of the males and females were injected with LHRHa.In addition,5 pairs of controls were set up without any hormone injection(wild type?×wild type?).The results showed that the spawning rate of wild type females which were paired with mutant and non-mutant males(42.9%)was little bit lower than control group(60%)in the first round of pair.The hatching rate of the fertilized eggs from the mutant males was significantly lower than control group(P<0.05).The spawning rate of mutant females was 25%,which was significantly lower than control group(60%).The mutant males(numbered 5-8)didn't show normal reproductive behaviors.It might be because of the knockout of cfGnRH gene in the female,which repressed the development of the gonad and ovulation.Also,the knockout of cfGnRH gene changed the reproductive behavior of mutant males,which couldn't give correct signal and trigger normal ovulation of wild type females.(4)Hormone test.In this study,3-year-old channel catfish P1 were used as materials,include 6 wild type fish,12 mutant fish and 8 non-mutant fish.Estradiol ELISA kits were utilized to test all the females.Testosterone ELISA kits were utilized to test all the males.GnRH ELISA kits were utilized to test both females and males.The results showed,for the fish which were not paired with other one,there was no significant difference of estradiol level and Gn RH level between mutant group and non-mutant group(P>0.05).,the average of estradiol level and GnRH level of mutant group were little bit higher than non-mutant group.For the fish,which were paired with other one without spawning,there was no significant difference among different groups(P>0.05),the average of estradiol level and GnRH level of the fish of wild type were little bit higher than mutant and non-mutant fish.For the fish,which were paired with other one with spawning,there was no significant difference among different groups,the average of testosterone level of the fish of wild type was little bit higher than mutant and non-mutant fish.This could be because of the mosaic phenomenon in the P1 generation and the exist of another type of GnRH of channel catfish.Therefore,these steroid hormones include estradiol and testosterone,might be regulated and released through other endocrine channels,and might be subject to negative feedback regulation,then the hormone level was increased in the experimental group.(5)Mutation assay of F1 generation.In this research,4 wild type females spawned,which were crossed with mutant and non-mutant males numbered 1-4.The mutant female numbered 9 which was crossed with 1 wild type male spawned.Then we got 5 families of F1 generation.The F1 population was used as materials.CEL-I mutation test was utilized to detect mutations.The results were compared with their mutant or non-mutant parents.It revealed that the gene mutations of F1 generation were consistent with the mutant male parents and female parents.The results from different parts of tissues were the same.Therefore,the fingerlings did not persist mosaic phenomenon,which indicated that F1 generation could be sterilized with more possibilities.