The Preliminary Research On Function Of Sox9b In Sex Determination And Differentiation In Medaka

Objective:Research on sex determination and differentiation regulation has important value and significance to revealing the complicatedly underlying molecular mechanism and promoting human disease research.Thus it has been appealing the attention of scientists,but genes in this system is the key for further exploration.Sex-determining systems in fish appear to be at a primitive stage of evolution.Medaka is the ideal model to study the molecular mechanism of the systems.Modern molecular biology techniques,especially gene editing technology,which have greatly promote the research on gene function.TALENs can identify the specific sequence and cut it down with exposure of DSBs,after micro-insertion or micro-deletion,ultimately cause frameshift mutation.Sox9b as a male-developmental factor,play a role in the early development of sex differentiation,but its function is undefined in Medaka.In this study,TALENs was applied to elucidate the function and mechanisms of Sox9b.Phenotype,fecundity data,histological observation and expression of related genes all provided experimental data and theoretical basis to the further research.Methods:To construct the TALENs-knockout plasmid,design knockout sites on targeted site.The linearized knockout plasmid were microinjected into the 1 cell stage fertilized eggs,mutation screening,established knockout families.The mutation detection,embryonic development,homozygous mutation detection,the morphological characteristics,secondary sexual characteristics,gonad index and reproductive statistics.The gonad tissue slice observation for exploring function of Sox9b.To explore the molecular mechanism with the semi quantitative or quantitative analysis.Results:1.TALENs-knockout plasmid work at the first exon of Sox9b,knockdown sites located in the upstream of the HMG box and C-terminal transactivation domain,and received 5 type of mutations.The mutation with 5bp base and 22bp base deletion were selected and established into mutation families.Two mutations encoded a truncated polypeptide with no functional domains.2.Mutant lines were cultured to F5,but no homozygote were found.Heterozygote ovulated less embryos and more abnormal embryos.Detection revealed a 1/2 proportion of homozygotes,significantly higher than that of 1/4 in normal,indicated that Sox9b homozygous mutation has a lethal effect.3.Detecting phenotype of mature heterozygote,sex-reverse did not occur,but the secondary sexual characteristics became atypical,GSI decreased significantly.Statistical analysis showed that heterozygous mutation had a negative impact on spawning,hatching rate,survival rate.According to the spawning amount,fertilization rate and survival rate,Sox9b mutation lead to fertility decline on female is more serious.4.Observation of gonadal tissue sections,ovarian structure obviously degenerated,almost no EVO and LVO,increased number of atretic follicles in Sox9b+/-Female.There was no significant structure change,but the cell number of all period significantly reduced in Sox9b+/-Male.5.The expression of sex related factors with semi quantitative detection,confirmed no sex-reverse in Sox9b++/-.The quantitative detection of sex related factors in Sox9b mutation found,female related factor Cypl9a1a,Foxl2 expression increased,male related factor DMY,Sox9b expression was significantly decreased,while Dmrtl,Amh the expression was significantly increased,the expression of Gsdf had no significant change.Conclusion:This study succeeded on Sox9b-knockout using TALENs technology and constructed Sox9b mutations.The Sox9b homozygote had lethal effect.In addition,the development of heterozygote was hindered,fecundity was also affected.There were rare EVO or LVO stage oocytes in the heterozygous ovary,and more atretic follicles.It implied that Sox9b played an important role in oocyte maturation.The quantitative expression indicated a competitive inhibition between Sox9b and Cypl9a1a,Foxl2.And there was positive feedback between DMY and Sox9b,yet,a negative feedback regulation between Sox9b and Dmrtl,Amh.Meanwhile,the regulation between Sox9b and Gsdf was distant or it had another compensatory regulation way.