Effection Of Temperature And 17β-estradiol On Sexual Differentiation Of Ictalurus Punctatus

In order to establish a systematic cultivation technology of pseudofemale channel catfish,this study establishes an accurate and rapid method to identify the genetic sex of channel catfish,peripheral primers,specific extension primer and DNA probe were designed according to a sex-linked SNP.The product of sex-linked SNP specific primer extension reaction was hybridized with DNA probe,and then detected by nucleic acid test paper based on immune colloidal gold technology,firstly.Secondly,this study aimed to explore appropriate 17β-estradiol doses and water temperature of feminization for male channel catfish fry before sex differentiation.Finally,genome-wide methylation sequencing and transcriptome sequencing were used to analyze differences in gene methylation levels and gene expression in transcriptome to further explore the role of genes in gender control.1.In order to establish an accurate and rapid method to identify the genetic sex of channel catfish,peripheral primers,specific extension primers and DNA probes were designed according to a sex-linked SNP.The product of nested PCR reaction was hybridized with DNA probe,and then detected by nucleic acid test paper based on immune colloidal gold technology.The results demonstrated that this method could accurately detect the genetic sex of channel catfish,which was consistent with the identification results of channel catfish sex-linked microsatellite markers.This study revealed that the specific primer extension reaction of sex-linked SNP combined with nucleic acid test paper based on immune colloidal gold technology could accurately and quickly identify the genetic sex of channel catfish.2.In order to explore the appropriate method of feminization and 17β-estradiol doses for male channel catfish,artemia and artificial compound feed wereas used as 17β-E₂ vector to feed catfish larvae continuously for 27 days.The growth data and survival rate of catfish in each group were measured and counted at 60 dahs,the sex reversal rate was also calculated in combination with the genetic sex identification results.Furthermore,the ovarian development of XX and XY females in each group was compared and analyzed based on the H&E staining of ovary tissue paraffin sections.Finally,17β-E₂ contents in gill,heart,liver,kidney,ovary and muscle tissues of sex XY females in each group were detected and compared with control XX females at 270 dahs.In addition,q RT-PCR was used to detect the expression levels of foxl2,dmrt1,dmrt2b,dmrt5 and dmrt6 in XX and XY female ovaries at 270 dahs.q RT-PCR was also used to detect the expression levels of gsdf gene in XY male testis,XX and XY female ovaries at 60 dahs and one and a half year old.The expression of gsdf gene in ovary of XX and XY female one and a half year old and testis of XY male fish was investigated by in situ hybridization of oligonucleotide probe m RNA.The results showed that the proportion of XY females and the number of oocytes in each group were increasing with 17β-E₂ doses.The weight and body length of female fish in each group first increased and then decreased with the increase of 17β-E₂ doses.The survival rate was no significant in each group.The content of 17β-E₂ in ovary was positively correlated with17β-E₂ doses,while no differences were observed in gill,heart,liver,kidney and muscle of female fish.The expression levels of foxl2 gene increased in XY females,while the expression of dmrt1 gene decreased in XY females.Compared with XX females,the expression of dmrt2b and dmrt5 in ovary of XY females were significantly up-regulated（P<0.05）,and the expression of dmrt1 and dmrt6 were significantly down-regulated（P<0.05）,suggesting that these dmrt gene family members may play an important role in the sex reversal process of XY females.The expression of gsdf gene in one and a half year old XY males was significantly different from that in XX and XY females（P<0.01）,but there was no significant difference in the expression of gsdf gene in 60 dahs old and one and a half year old XX females and XY females（P>0.05）.In situ hybridization results showed that gsdf m RNA was expressed in sertoli cells of testis,but not in ovaries of normal females,and only a small amount in ovaries of XY females.The results indicated that gsdf gene was mainly involved in the maintenance of testis.This study established 17β-E₂ induced feminization of XY male channel catfish,and laid a solid foundation for the subsequent cultivation of new male channel catfish varieties.3.This study aimed to explore appropriate water temperature of feminization for male channel catfish fry before sex differentiation.The growth data,survival rate and ovarian formation ratio of fish in each treatment group were measured and counted at 60 dahs,the sex reversal rate was also calculated in combination with the results of genetic sex identification.The ovarian development of XX and XY females in each group was compared and analyzed based on the anatomy and H&E staining sections.Finally,study further on normal temperature induced XY females and genome-wide methylation sequencing,through the analysis of the methylation of each sample type,the level of methylation and methylation analysis differences of genes,the expression of the differences in gene transcription group analysis,at the same time the transcriptome expression quantity analysis of gender related gene.The results showed that the development of oocytes in the ovary of XY female accelerated with the increasement of temperature.The survival rate gradually decreased,while the body length and weight first increased and then decreased.The whole genome methylation sequencing of normal female fish and temperature induced XY female fish was carried out.In order to analyze the different methylation level genes,firstly,the methylation type and methylation level of each sample were analyzed.Secondly,the expression of differential genes was searched in the transcriptome,at the same time,the transcriptome expression of gender related factors was analyzed,and the gender regulation pathway of channel catfish was further researched.It was found that genetic variations of steroid hormone biosynthesis pathway enrichment of signaling pathways by analyzing differences in gene transcription promoter regions.XY female fish female gender related genes are activated in the transcriptome expression,male related genes have been suppressed,XY female fish and XX female fish gender related gene expression of no significant difference in the transcriptome.The study realized the feminization of male channel catfish by using temperature induction-based method,which could provide a kind of environment-friendly sex control breeding method for channel catfish.In addition,this study provides a theory for further study on the mechanism of temperature-induced feminization of channel catfish.