| Hyriopsis cumingii is a unique freshwater pearl mussel in our country.It has a very important position in the freshwater pearl industry.In the actual breeding process,males have better pearl production performance than females.Therefore,research on sex determination and sex differentiation is very important for the practice of H.cumingii.The research on the related mechanisms of sex determination and sex differentiation and sex determination genes of higher animals is relatively complete,but H.cumingii is invertebrate animal,and it lacks heterotypic sex chromosomes,the mechanism of sex determination and sex differentiation is still unclear.Therefore,studying the genes related to sex determination of Hyriopsis cumingii has important theoretical and practical value.In this experiment,the full length of the 3 sex-difference genes named as kinase X,Calm and Mlck.There were cloned by the RACE method,and the sequence characteristics of these 3genes were analyzed.Fluorescence quantitative method was used to analyze the expression level of 6 tissues of sexually mature individuals and early gonad expression,in situ hybridization was used to study the expression positions of these three genes.RNA interference technology was used to explore the upstream and downstream relationship between the kinase X gene and Sox9 gene in the c AMP pathway and the upstream and downstream relationship between Calm gene and Mlck gene in oxytocin pathway.Through the above methods,the role of these 3 genes in the sex determination and sex differentiation of H.cumingii was preliminarily explored.The specific research results are as follows:1.Molecular identification and functional study of the kinase X gene in H.cumingiiThe full length of kinase X gene is 1652 bp,of which the 3’UTR is 314 bp,the 5’UTR is 45 bp,the open reading frame(ORF region)is 1293 bp,and 430 amino acids are predicted.kinase X gene is expressed in 6 tissues,including foot,gill,mantle,gonad,adductor muscle,liver.The highest expression tissue is the male gonad,and the expression of females in each tissue are high than that of male;in early gonadal tissues,the expression of kinase X gene at 6 months was significantly higher than that of other months;in gonad of12、24 and 36 months,the expression of gonads at 24 months was significantly higher than that of 12 and 36 years of age.It is speculated that the kinase X gene plays a certain biological role in the process of male sexual maturation.In situ hybridization results showed that kinase X gene was not only expressed on the follicular membrane in female gonads,but also expressed on the egg cell membrane.While in male gonads,it is expressed in spermatogonia and spermatocytes.After interference of the kinase X gene,it was found that the expression of Sox9 gene was also falling,and the synthesized interference chain was effective.Among them,the most effective interference chain had an interference rate of 74.58% for the kinase X gene in the female gonads,and 96.53% in males.Based on this,it is speculated that kinase X gene is related to male sex development,and the interference rate of this interference chain on the Sox9 gene in female gonads reached 90.3% in females and 56.6% in males,The results showed that SOX9 was downstream of the kinase X gene in the c AMP pathway.2.Molecular identification and functional study of the Calm gene in H.cumingiiThe full length of Calm gene is 1144 bp,of which the 3’UTR is 527 bp,the 5’UTR is167 bp,the open reading frame(ORF region)is 450 bp,the predicted amino acid is 149.Calm gene is expressed in 6 tissues including foot,gills,mantle,gonad,adductor muscle,liver,among which the expression level of female gonads is higher than that of male gonads;in early gonad,the expression level of Calm gene at 1 month is the highest,it rises at 7 months,and then drops to a normal level;the gonad fluorescence quantitative results of 12,24,and 36 months of age show that the expression of Calm gene in females is higher than that of males,and the difference between male and female is extremely significant,of which 36 months gonad has the highest expression level.The results of in situ hybridization show that Calm gene is not only in female gonads.It is expressed on the follicular membrane and the egg cell membrane,while in spermatogonia and spermatocytes in male gonads.The pre-experiment screened out the interference chain with the highest interference rate,and then expanded the number of sample to interfere with the Calm gene.The interference rate in female gonads reached 61.04%,and the interference rate in males reached 51.05%.Since the interference rate in females is higher than that in males,it is speculated that the Calm gene is related to female sex development.3.Molecular identification and functional study of the Mlck gene of H.cumingiiThe full length of Mlck gene is 1260 bp,of which the 3’UTR is 482 bp in length,the5’UTR is 13 bp in length,the open reading frame(ORF region)is 765 bp,and there are 254 predicted amino acids.The Mlck gene is expressed in 6 tissues,including foot,gill,mantle,gonad,adductor muscle,and liver.Among them,the tissue with the highest expression is the female gonad,and the expression of female is all higher than that of males,in the early gonad,the Mlck gene expression level is the highest at 8 months,and the expression level is lower at 6 and7 months;in the gonads at 12,24,and 36 months,females at 24 months is higher than males,but at 12 months and 36 months,males are higher than females.In situ hybridization results show that the Mlck gene is expressed in the follicular membrane and egg cell membrane of females.In males,the signal is mainly located on spermatogonia and spermatocytes.After interference with the Calm gene,it was found that the expression of Mlck gene was also falling,and the interference rate in female gonads was 27.17%,while the interference rate in males was 31.36%.There is a gene interaction relationship between Calm gene and Mlck gene.The expression of Calm gene promotes the expression of Mlck gene,but compared with the expression of Sox9 gene after kinase X gene interference,the correlation between Calm gene and Mlck gene is not strong. |
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