| Kdm6b is a histone demethylase,and in fish,due to the duplication of the genome during evolution,the Two homologous genes exist in Kdm6b:Kdm6ba and Kdm6bb.Previous studies in our laboratory found that:there is variable splicing phenomenon in Kdm6bb,which produces two transcripts:Kdm6bb＿tv1（intron 5 cut）and Kdm6bb＿tv2（intron 5 retained）,and the expression levels of the two transcripts differ significantly between males and females,and Kdm6bb＿tv1 was mainly expressed in sexually differentiated males,while Kdm6bb＿tv2 was mainly expressed in sexually differentiated females,suggesting that Kdm6bb may play an important role in the sexual differentiation of Nile tilapia.Therefore,it is significant to reveal the important roles of two variable splice transcripts of Kdm6bb in the sex differentiation process of Nile tilapia and their mechanisms of action.In this study,we investigated the role of two variable splice transcripts in the sex differentiation of Nile tilapia by overexpression and CRISPR/Cas9 knockdown,in order to provide a theoretical basis for the breeding of all-male tilapia fry.The specific experimental results are as follows:1.Construction and screening of Kdm6bb＿tv1 and Kdm6bb＿tv2 overexpression individuals.The recombinant plasmids Ef1α:Kdm6bb＿tv1-Myc and Ef1α:Kdm6bb＿tv2-Gfp were successfully constructed by PCR amplification of Kdm6bb＿tv1 and Kdm6bb＿tv2 CDS region sequences and inserted into p Tol2 vector,respectively.The F0 generation positive individuals of Kdm6bb＿tv1 overexpressing fish were screened according to RT-PCR and Western blot,the F1 generation individuals were generated from the F0 generation positive individuals and WT individuals,and the F1 generation positive individuals were mated to generate F2 individuals as the Kdm6bb＿tv1 overexpression group for subsequent experiments;similarly,the Kdm6bb＿tv2 The Kdm6bb＿tv2 F2 generation overexpression group was sequentially obtained by screening positive Kdm6bb＿tv2 overexpression individuals based on GFP fluorescence expression for subsequent experiments.2.Effects of overexpression of Kdm6bb＿tv1 or Kdm6bb＿tv2 on early sex differentiation and gonadal development.（1）KDM6BB＿TV1 protein expression in F2 generation individuals overexpressing Kdm6bb＿tv1.Immunohistochemical results showed that at 30 dpf,a strong positive signal of KDM6BB＿TV1 protein was detected in the gonads of Kdm6bb＿tv1 overexpressing XX individuals,which was significantly higher than that of control females and similar to control males;at 60 dpf,the expression level of KDM6BB＿TV1 protein further increased and could be detected in Leydig cells and some other somatic cells was detected in Leydig cells and some other somatic cells.（2）Effect of overexpression of Kdm6bb＿tv1 or Kdm6bb＿tv2 on sex differentiation.Immunohistochemical results showed that at 30 dpf,positive signals for DMRT1 protein were detected in Kdm6bb＿tv1 overexpressing XX individual gonadal support cells,but not for CYP19A1A protein;positive signals for CYP19A1A protein were detected in Kdm6bb＿tv2overexpressing XX individual gonadal mesenchymal cells,but not for CYP19A1A protein.positive signal of DMRT1 protein was detected.（3）Effect of overexpression of Kdm6bb＿tv1 or Kdm6bb＿tv2 on gonad development.HE results showed that at 30 dpf,the gonad morphology of Kdm6bb＿tv1 overexpressing XX individuals was similar to that of control males,and Kdm6bb＿tv2 overexpressing XX individuals was similar to that of control females;at 60 dpf,the gonads of Kdm6bb＿tv1overexpressing XX individuals could be clearly identified in the gonads of control females.At60 dpf,spermatocytes and spermatogonia could be clearly observed in the gonads of Kdm6bb＿tv1 overexpressing XX individuals;oocytes and oogenic cells could be clearly observed in the gonads of Kdm6bb＿tv2 overexpressing individuals.3.Effect of overexpression of Kdm6bb＿tv1 or Kdm6bb＿tv2 on sex hormone levels.ELISA results showed that at 90 dpf,serum levels of 11-KT were significantly up-regulated and E2levels were significantly down-regulated in Kdm6bb＿tv1 overexpressing XX individuals compared to normal females,similar to normal males;while serum levels of both 11-KT and E2 were largely unchanged in Kdm6bb＿tv2 overexpressing XX individuals4.The effect of overexpression of Kdm6bb＿tv1 or Kdm6bb＿tv2 on the sex reversal rate of Nile tilapia females.The statistical results showed that the sex reversal rate of females in the control female group was 23.5%,while the average sex reversal rate of females in the F2 of Kdm6bb＿tv1 overexpression group was 61%,which was significantly higher than that of the control female group;the average sex reversal rate of females in the F2 of Kdm6bb＿tv2overexpression group was only 22.5%,which was similar to that of the control female group.5.Construction of Kdm6bb＿tv1 knockout individuals and the effect of knockout Kdm6bb＿tv1 on the sex reversal rate of Nile tilapia females.Kdm6bb＿tv1 was knocked down using CRISPR/Cas9 technology,F0 generation positive individuals were screened using PCR,F1 generation individuals were generated from F0 generation positive individuals and WT individuals,and F1 generation positive individuals were mated to generate F2 individuals for subsequent experiments.The results showed that（1）all Kdm6bb＿tv1-/-pure knockout individuals died by 55 hpf of development,and only Kdm6bb＿tv1+/-heterozygous knockout individuals were obtained.（2）The mean sex reversal rate of Kdm6bb＿tv1+/-heterozygous knockout females was 22.7%using the gonadal press method,which was similar to that of control females.In summary,overexpression of Kdm6bb＿tv1 up-regulated the expression of Dmrt1,a key gene for male sex differentiation,and down-regulated Cyp19a1a,a key gene for female sex differentiation;up-regulated serum levels of 11-KT and down-regulated serum levels of E2;and significantly increased the sex reversal rate in XX females.Overexpression of Kdm6bb＿tv2had no significant effect on the expression of sex differentiation key genes,serum levels of sex hormones and sex reversal rate in XX females.Km6bb＿tv1+/-heterozygous knockout had no significant effect on sex reversal rate in females. |
