Histological Observation And Cloning And Expression Analysis Of Dmrt1 Genes In The Development Of Spotted Knifejaw Gonad

In order to study the gonad development characteristics of knifejaw gonad(Oplegnathus punctatus)and the expression of dmrt1 in the process of gonad differentiation and development.This study will be elaborated from two aspects: one is the method of using histology slice research spot stone bream gonadal differentiation and development process,the second is gender related gene cloning for dmrt1 open reading frame(ORF),and through the fluorescence quantitative PCR analysis of the gene in spot stone bream fish of different organizations and in the larval expression in different development stage,to explore the function of the gene.One,as the research spot stone bream process,the differentiation of gonads conventional paraffin tissue section method is used to observe spot stone bream gonad development and differentiation,the results show that the spot for stone bream gonad development type,unisexual testis belongs to the lobular type,120 days of age in vas deferens primordium,testis differentiation,180 days of age began to form fine lobular marked complete,the differentiation of testis anatomy and simultaneous primary spermatocyte,marked the beginning of testis cytological differentiation;At the age of90 days,the primitive gonads began to develop into clusters of cells,which later developed into ovaries,marking the beginning of ovarian anatomical differentiation.At the age of 150 days,the embryonic form of the ovarian cavity appeared,and at the age of 180 days,the formation of primary oocytes marked the beginning of ovarian cytological differentiation.It can be seen that the female porcine is anatomically differentiated earlier than the male,and the male and female are approximately the same in cytological differentiation.Two,the spot stone bream testis development can be divided into six periods,the occurrence of sperm can be divided into five stages: 60-90 days of age for Ⅰ testis,exterior line is long and thin and so on,can’t distinguish between male and female;Before and after 180 days of age for Ⅱ testis,the appearance of a flat belt,spermatagonial cells began rapidly dividing,this time in the testis of the primary spermatocyte;210-240 days of age for Ⅲ testis,macroscopic observation to the testis reddish rod,can distinguish between male and female,the period,mainly primary spermatocyte and began the meiosis produces secondary spermatocyte;460 days of age for Ⅳ testis,appearance has milk-white rod-shaped,lobular cavity in a small number of sperm cells,squeeze the abdomen without semen out,24 months of age for testis Ⅴ appearance,white color block,in a large number of fine utricle and vas deferens sperm,gently press abdomen has a small amount of semen out,26 months of age for Ⅵ testis,atrophy of testis volume small,dark yellow,sperm degenerationabsorption.The spermatogenesis in the process of testis development is obviously different from that in the process of testis development,which determines that spermatozoa mature gradually.Ovarian development is divided into six periods,is divided into six phases during oocyte development,when the first Ⅰ oocytes oogonium stage,when the first Ⅱ phase oocytes significantly larger volume,entered the stage of oocyte niche for a long time,the first phase oocytes early appear when Ⅲ nucleoli,mid and late to rise in the Numbers,and follicular cells,when the first Ⅳ phase is oocytes in large growing phase of the cell,the cell volume increased obviously,yolk granules increase,when the first Ⅴ oocyte nucleus dissolved,nucleolus disappeared,the entire cell is full of yolk,the first Ⅵ phase oocytes began to shrink.Three,by homology cloning spot stone bream dmrt1 gene open reading frame(ORF),the open reading frame 885 bp long genes,encoding 294 amino acids,by fluorescence quantitative PCR assay dmrt1 genes in spot stone bream fish different expression levels in the organization,and found that dmrt1 gene expression is dominant in the testis,in other organizations(ovaries,liver,spleen,kidney,muscle,brain,gill wire,intestine,heart,etc.)in expression,not even express,dmrt1 genes associated with male gonad development.By measuring dmrt1 genes in spot stone bream larvae different development period(150 d,180 d,210 d,240 d,270 d),the expression level of,found in the male adolescent spot stone bream 210 d is used to express quantity is less,210 d expression peak,expressing quantity showed a trend of decline after 210 d,in every period of female juvenile spot stone bream from trace to express,don’t even express.Gonadal section experiments showed that early sperm cells appeared in young male porphyrians at day 210 d,suggesting that dmrt1 plays a regulatory role in the meiosis of sperm cells and the formation of sperm cells in the testis,and is related to the maintenance of the testis.