The Study Of Gonadal Differentiation Process And Sex-related Gene Sox3,sox9 Of Spotted Halibut

Spotted Halibut(Verasper variegatus)belong to Pleuronectiformes(Pleuronectiformes),Pleuronectidae(Pleuronectidae),star sole(Verasper).It is a marine flatfish species which has an important commercial value.There were significant differences in growth rate and body size between the male and female spotted halibut,and the females were significantly larger than the males.All female cultivation can improve breeding efficiency.Researches on mechanism of sex determination and features of sex differentiation could provide valuable theoretical basis for all female cultivation.We successfully identified the sox3 genes and sox9 genes of spotted halibut by RACE,explored the dynamic expression patterns of sox3 and sox9 in larvae and adult by q PCR,observed the differentiation process of sexual gland by histological sectioning.This study would provide basic information for the sex determination and gonadal development mechanism of spotted halibutExperiment 1 Gonadal differentiation of spotted halibut was investigated by histological sectioning.the results are as follows:(1)On the 30th-35 th day post hatching(dph)(the total length reached 15.5-17.4 mm),primitive germ cells appeared in primitive gonad.They were bigger than somatic cells.Their nucleus were large,and nuclear membrane were cleaar.On the 40th-55 th dph(the total length reached 19.6-32.8 mm),the length of gonad primordium increased significantly,while the width didn't change very much,The germ cells proliferated rapidly,multiple germ cells were clearly visible from the transverse section of the gonads,inlaid in the gonads.(2)Ovarian cavity firstly appeared at 60 dph(the total length reached 36.2mm),hyperplasia at one end of the gonad and extending to the other end;On the 70 th dph(the total length reached 45.1 mm),the sex gland begins to proliferate from one end,extend to the other end,the ovarian cavity further expands.Female fish of 80 th dph,the total length reached 57.7mm,the ovarian cavity was fully formed.The ovary cells proliferated rapidly along the spawning plate and the ovaries reached a complete differentiation;A few oocytes in the ovaries were found in females on 90 th dph.Oocyte nuclei were large,occupying almost the entire oocyte,1-3nucleoli.(3)There was no obvious differentiation characteristic in the gonads of 70 th dph males.The gonad of 70th-80 th dph males began to proliferate from one end,and extended to the other end,the volume increased further,the spermatogenic cell began to proliferate rapidly.Conclusion: Ovarian cavity firstly appeared at 60 dph,which indicated that two types of gonads were distinguishable at 55-60 dph.Experiment 2 We successfully identified the sox3 genes of spotted halibut.The total length is 1723 bp,including 891 bp ORF,encoding 297 amino acids,5 'UTR is 48 bp and 3' UTR is 784 bp.There are polyadenylic acid tails and caudal signals AATAAA in 3 'UTR.The expression of sox3 gene were different in brain,eye,gills,heart,liver,bravery,bowel,testis,kidney and muscle in spotted halibut.The transcription levels were high in gills and ovary,especially in ovary which was significantly higher than other tissues.Gender two phase difference could be observed in gonads,the expression level of sox3 gene in ovary was significantly higher than that in testis,indicating that sox3 gene might associate with female gonad development.The dynamic expression patterns of sox3 gene in spotted halibut larvae showed a gradually decline from 20 to 50 days after hatching and a sudden rise after 50 days.And through the gonad slice experiment,we can found larval gonads begin to differentiate from 55 to 60 days,this expression level rise might associate with larval gonad differentiation.sox3 gene may be involved in larval gonadal differentiation process.Experiment 3 We successfully identified the sox9 genes of spotted halibut.The total length is 3287 bp,including 1431 bp ORF,encoding 477 amino acids,5 'UTR is 368 bp and 3' UTR is 1488 bp.There are polyadenylic acid tails and caudal signals AATAAA in 3 'UTR.The expression of sox9 gene were different in brain,eye,gills,heart,liver,bravery,bowel,testis,ovary,kidney and muscle in spotted halibut.The transcription levels were high in gill,brain and testis,especially in testis which was significantly higher than other tissues.Gender two phase difference could be observed in gonads,the expression level of sox9 gene in testis was significantly higher than that in ovary,indicating that sox9 gene might associate with male gonad development.The dynamic expression patterns of sox9 gene in spotted halibut larvae showed the level of expression was not significant.