Phenotypic Analysis Of Transgenic Zebrafish With The Nitroreductase Expression Under The Control Of Zebrafish Gsdf Gene Promoter

With the rapid development of transgenic technology,many studies have begun to use transgenic technology to construct and cultivate cultured fish with excellent traits.Considering that genetically modified individuals may escape into the natural environment,causing a series of ecological risks.Therefore,it is difficult to promote the demonstration of genetically modified fish.Cultivating sterile transgenic lines is an effective way to solve this problem.Hybrid sterility technology and triploid sterility technology were traditional transgenic technologies to induce sterility,the above methods are reasonable to some extent,but they also have some disadvantages,it is difficult to breed fish on a large scale and achieve complete sterility.In recent years,many studies have begun to apply transgenic and gene editing techniques to construct infertile individuals.Compared with traditional fish sterility technology,transgenic technology can be used to generate large-scale transgenic fish,and the offspring can be complete sterility theoretically.Therefore,this technology will become an important method to breed sterile fish in the future.The gonads of fish are composed of germ cells and somatic cells.At present,transgenic or gene editing techniques are used to generate sterile fish,and most studies focuse on germ cells,such as inducing their apoptosis or knocking out their growthrelated genes,few studies focuse on somatic cells to carry out related research.Gonadal somatic cell derived factor(gsdf)is a new member of the transforming growth factor beta(TGF-β)superfamily and is only present in teleost fish,specifically expressed in somatic cells of the gonads.Nitroreductase B(nfsB)gene encoded Nitroreductase(NTR)protein combines with Metronidazole(Mtz)to produce a cytotoxic active substance,which eventually leads to cell death.In the present study,we used zebrafish(Danio rerio)as a model and used the Tol2 transposon technology to construct a transgenic line,in which NTR can be specifically expressed in gonadal somatic cells by transgenic technology.Then the transgenic line was exposed with Mtz to induce cell ablation.The main results are as follows:1.We cloned the core promoter region of the zebrafish gsdf gene,the total length of which is 2045 bp.We inserted gsdf promoter into the pmini-nfsB-m Cherry plasmid to obtain pmini-gsdf-nfsB-mCherry plasmid.Tol2 transposase mRNA and pmini-gsdfnfsB-mCherry plasmid were injected into wild-type F0 fertilized eggs at the 1-2 cell stage.The F0 generation zebrafish were screened out by PCR detection and gene sequencing,and then outcrossed with the wild-type zebrafish to get the F1 heterozygous zebrafish.PCR detection and gene sequencing were used to screen out the foreign gene fragment nfsB-mCherry from the F0 generation,and F0 positive generation zebrafish were outcrossed with the wild-type zebrafish to get the F1 heterozygous zebrafish.RTPCR results showed that F1 generation individuals began to express nfsB-mCherry at 6 dpf.In sexually mature F1 individuals,the mRNA of nfsB-mCherry and mCherry fluorescence signal were only expressed in the gonads.The F1 generation could stably inherit the foreign gene fragment nfsB-mCherry to the offspring.2.The F1 generation transgenic zebrafish juveniles were exposed to 5 mM Mtz starting from 21 dpf and 28 dpf,exposed for 14 and 21 days respectively,all individuals were withdrawn Mtz exposure until 42 dpf and raised up to sexually mature(5 months old).At the same time,two control groups were set up,the group one,the transgenic juveniles exposed to DMSO(Mtz solvent),and the group two,the wild-type juveniles exposed to 5 mM Mtz.The results from sex ratio of sexually mature zebrafish showed that after transgenic juveniles were exposed to Mtz for 14 days and 21 days,the percentage of males were 100%and 76%respectively;after transgenic juveniles were exposed to DMSO in control group,the percentage of males were 63%and 41%respectively;after wild-type juveniles were exposed to Mtz for 14 days and 21 days in the other control group,the percentage of males were 90%and 88%respectively.We further analyzed the fertility of male fish,there was no significant difference in GSI and fertilization rate between the males of the experimental group and the males of the two types of control groups in the 14-day Mtz exposure group,and the GSI and fertilization rate of male in the 21-day Mtz exposure group were significantly lower than those of the other two control groups(P<0.05).Histological results showed that the testis structure of the sexually mature male fish in the experimental group was normal in the 14-day Mtz exposure group;in the 21-day Mtz exposure group,the diameter of the seminiferous tubules of sexually mature males in the experimental group was relatively small,the number of sperm was significantly less.Analysis of the female’s fertility(total egg production within 12 days,GSI)showed that no significant differences between the experimental group and the control group.3.After sexually mature transgenic and wild-type zebrafish were exposed to 5 mM Mtz for 14 days,the GSI of transgenic male fish was significantly lower than that of wild-type(P<0.01),but there were no significant differences in GSI between transgenic and wild-type female fish.The fertility test results showed that the fertilization rate of transgenic male fish was significantly lower than that of wild-type male fish(P<0.05);and the total number of total egg production within 12 days was significantly lower than that of wild-type female fish(P<0.001).Further histological analysis showed that the number of sperm cells in each stage of transgenic male fish testis was significantly lower than that of wild-type testis;transgenic and wild-type female fish lacked mature stage oocytes in their ovaries.40 days after the end of Mtz exposure,the GSI of the male and female fish in the experimental group and the control group increased with no significant differences;the testis structure of the transgenic fish and wild-type fish was normal and there was no obvious difference,the ovary structure of the transgenic fish and the wild-type fish was normal and there was no obvious difference.4.The real-time qPCR was used to analyze the expression of gsdf,piwil,dazl,sycp3l in the transgenic zebrafish testis after 5 days Mtz exposure and the expression of gsdf and cypl9ala in the ovary after 14 days Mtz exposure,and the results showed that expressions of gsdf,piwil,dazl,sycp3l did not decrease significantly,and the expression of gsdf and cypl9a1a in females decreased significantly(P<0.05).In summary,we generated a transgenic zebrafish line that can be conditionally induced the ablation of somatic cells by exposing Mtz,which can significantly affect the fertility of zebrafish.This study has certain reference significance for the related research of fish reproduction,sex control,gonad regeneration and so on.