| Little yellow croaker Larimichthys polyactis is one of the most important marine economic fish in China.At present,the artificial breeding of little yellow croaker is successful.From artificial breeding,that females are larger in size than males,and that full-female breeding has higher economic benefits.Also,little yellow croaker has a strong stress response during breeding,which can lead to injury or death of the fish.In this study,different concentrations of 17?-estradiol?E2?were used to expose in cultured water of juvenile little yellow croaker,and the effects of this hormone on growth and gonadal development of little yellow croaker were studied by routine measurement and tissue section observation.And bioinformatics analysis of the key enzyme genes dbh and pnmt,which are adrenaline synthesis step of the sympathetico-chromaffin system in the stress response.The expression of dbh and pnmt genes in brain and kidney of little yellow croaker was studied using qRT-PCR under the treatment of sound and light stress.The results are as follows:1.E2 exposure experimental groups were significantly lower than the control group on the body length and weight in 20-60 dat?days after treatment?.Additional,E2 exposure experimental groups were no significant difference from female control group C?0?.GSI?Gonadosomatic index?level in 60 dat.Morphology and histology observation showed that the E2 exposure concentration 1 and 10?g/L groups gonads became stubby in head end and appeared 2 to 3 phase of oocytes with obvious ovarian cavity,part of 50?g/L group gonads were development deformity.Testis and undifferentiated gonad were not found when concentration of E2 above 10?g/L,and the rate of deformity increased with the concentration of E2.The sex ratio of E2 treated groups concentrations 1,10 and 50?g/L were 82%,88%and 63%respectively,and the control groups were approximately 1:1.2.One dbh and two pnmt genes?pnmt a,pnmt b?were identified by splicing and identification from the whole genome of the little yellow croaker.The dbh gene contains12 exons and 10865 bp in length,The coding region is 1859 bp.A total of 605aa protein sequence,pnmt a contain 3 exons,the full-length 2056bp,the coding region is 822 bp,a total of 180aa protein sequence,pnmt b contains 3 exons,a total length of 2908bp,a total of 543bp coding region,a total of 180aa protein sequence.The amino acid sequence was more than 65%homologous to that of fish,and the closest relationship was found with the large yellow croaker Lrimichthys crocea.From the transcriptome data,the dbh gene is expressed in the intestine,brain,kidney,head kidney,and testis,and is slightly expressed in the skin,gills,eyes,ovary,and spleen.The pnmt a gene is highly expressed in brain,kidney and head kidney tissues,and slightly express in the intestine,skin,brain,gills,and testis.pnmt b is expressed in the intestine,skin,gills,brain,kidney,head kidney,and testis.slightly express in the liver,eyes,heart and spleen.3.By detecting the expression of pnmt a,pnmt b,and dbh in brain and kidney tissues at different times 15s,90s,and 300s.In the brain tissue stimulated by light,the expression levels of pnmt a and dbh genes in the 300s group were the highest in the same experimental group,and the expression level of pnmt b gene in the 90s group was the highest in the experimental group.In kidney tissue,the expression levels of pnmt a,b,and dbh in the 300s group were the highest in the experimental group.In the brain tissue stimulated by sound,the expression levels of pnmt a,b,and dbh in the 300s group were the highest in the experimental group.In kidney tissue,the expression level of pnmt a gene in the 15s group was the highest in the same group,and the expression levels of pnmt b and dbh genes in the 300s group were the highest in the experimental group.light and sound can arise the sympathetico-chromaffin system of small yellow croakers,which can lead to the upregulation of dbh and pnmt a,b genes in small yellow croakers,also the expression levels increase with time.In this study,E2 was used to induce little yellow croakers.When E2 concentration was 10?g/L,the induction effect was the best,mainly in the highest female rate.It laid the theory to study the mechanism of sex differentiation and the possibility of full female breeding in the little yellow croaker.Bioinformatics analysis of dbh and pnmt genes of little yellow croakers revealed that dbh and pnmt genes are closely related to large yellow croaker.The pnmt gene may have a replication gene.Under the stress and light stress treatment,the gene expression of dbh and pnmt genes in brain and kidney tissues of the little yellow croaker was upregulated,which laid the foundation for further study of the stress response mechanism of fish. |
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