The Establishment Of Genome Editing Technique (TALEN) And Functional Analysis Of Male Sexdetermining Gene Dmrt1 In Chinese Tongue Sole (Cynoglossus Semilaevis)

1.Genome editing techniques(TALEN)in Chinese tongue soleGenome editing technology has great significance and application value for gene functional analysis and genetic engineering breeding in marine animals.It is very important to carry out genome editing technology of marine aquaculture animals,which is very important for cultivating new varieties of marine aquaculture.However,because of the difficulty of microinjection technology of mariculture fish embryo,coupled with the small number of marine fish larvae and difficult to cultivate survival and other issues,seriously hampered the mariculture fish genome editing technology process,so far There is no report on the success of the genome editing of mariculture fish at home and abroad.In this study,the microinjection technique and genome editing technique of Chinese tongue sole are successfully established,and the breakthrough of gene function was confirmed.The GFP plasmid was successfully microinjected into the embryos of Chinese tongue sole and expressed.The dmrt1 gene on Z chromosome was successfully knocked out using TALEN technique,33 mutated fish were obtained.2.The functional analysis of dmrt1 associated with sex determination and gonadal development in Chinese tongue soleHere we show that microinjection of TALEN m RNAs targeted towards dmrt1 into sole embryos efficiently induced mutations of this gene with high frequencies up to almost 100%.The ZZ dmrt1 mutant fish developed ovary-like testes with a central cavity,and the spermatogenesis process was disrupted.The female sex fate related genes foxl2 and cyp19a1 a were significantly increased in the gonad of the ZZ dmrt1 mutant.Similar to the control ZW female,the dmrt1 deficient ZZ fish grew much faster than the ZZ male control.Notably,we obtained a hermaphrodite ZW fish with a testis on one side and an ovary on the other side.This fish was chimeric for a dmrt1 mutation in the ovary,and wild-type dmrt1 without mutation in the testis.Taking together,our data provide the first functional evidence that the Z-linked dmrt1 is a male sexdetermining gene in tongue sole and that this gene is linked to the distinct growth dimorphism between the two sexes of C.semilaevis.3.The establishment of male sex-determining gene dmrt1 mutant fish family in Chinese tongue soleThe dmrt1 gene was knocked out by genome editing technique in Chinese tongue sole,and the dmrt1 mutant fish were cultured to sexual maturity.And then in 2015 and 2016,the dmrt1 gene mutation of the F0 generation ZZ male and wild female,mutant F0 generation ZW female and wild male,mutant F0 on behalf of the female Fish and male were crossed to obtain hybrid offspring fish by artificial breeding technology,and the detection of dmrt1 gene of offspring was carried out.The results show that the parental dmrt1 mutant gene is inherited to the offspring.In the future,further artificial breeding work will be done,and eventually get fast growing family.