| Parental care is common in mammals and allows offspring to obtain milk,a substance rich in a range of nutritional and non-nutritional factors crucial to the survival of a developing newborn.The discus fish Symphysodon spp.,an Amazonian cichlid,show unusual behaviour: free-swimming fry bite the skin mucus of their parents’ for growth and development 1 month after hatching.This is similar to the breastfeeding behaviour of mammals.However,little is known about the mechanism by which discus secrete ’milk’ and the related genes involved.Therefore,transcriptome sequencing was performed using the brain tissues and skin tissues of female discus during the parental and non-parental periods.By analysing the brain transcriptome,Through the comparison between parents and non-parents,86 differentially expressed genes were obtained,among which 71 genes with an elevated expression trend and 15 genes with a downward expression trend were found.Including up-regulated nrif2,ednrb,laptm,foxb,and otx2 but down-regulated prkcd,h1-5,sox1,and hbe.Through KEGG enrichment analysis,20 different gene metabolism pathways were found,including regulation of TRP channel by inflammatory mediators,vascular smooth muscle contraction,neurotrophic signalling pathway.In skin transcriptome analysis,228 differentially expressed genes were obtained,among which 126 genes with an elevated expression trend and 102 genes with a downward expression trend were found.mrc,itga11 and ptprj but down-regulated junb,dpp3,casp3,ppid,aloxe3,hbe and gale during parental care.By KEGG pathway analysis,seven enriched pathways were obtained during discus parental care,including arachidonic acid metabolism,adhesion,apoptosis,steroid biosynthesis,tuberculosis,P53 signaling and serotonergic synapse.In order to study the role of otx2 gene in parental care of discus,the otx2 gene of discus was cloned by RT-PRC.the se quence length was 876 bp,290 amino acids were encoded,the predicted molecular weight was 31771.55 da,and the theoretic al isoelectric point was 9.40.smart,Swiss-Model and MEGA software were used to analyze the structure of genes and the evolutionary relationship of species.The amino acid sequence prediction showed that the gene had an otx conserved domain and was closely related to Oreochromis niloticus and Myripristis murdjan in terms of evolutionary relationship.According to the homology analysis.The results showed that the gene had more than 75% homology with the amino acid sequence of Oreochromis niloticus,Echeneis naucrates,Salarias fasciatus,Paralichthys olivaceus and Myripristis murdjan.q-PCR analysis of spatial expression fluorescence found that otx2 gene was expressed in brain of female discus,heart,liver,kidney and other tissues,and the expression was significantly high in the brain.In situ hybridizations showed that otx2 gene was expressed in periglobular gray matter zone,vertical pillow,and parietal compartment of the middle brain of discus.In order to investigate the role of mrc gene in the parental care process of discus,mrc gene was cloned by RT-PCR.The length of c DNA region of mrc gene was 876 bp,encoding 291 amino acids,the predicted molecular weight was 33440.42 Da,and the theoretical isoelectric point was 5.39.In terms of evolutionary relationship,it was compared with the multiple sequences of the mrc protein of vertebrates such as Maylandia Zebra,Oreochromis niloticus,Astatotilapia calliptera,Neolamprologus brichardi,Channa argus,Labeo rohita,Fundulus heteroclius.The similarity of mrc amino acid sequence of the discus was lower than 75% with that of other animals.Quantitative PCR spatial expression analysis showed that mrc gene was expressed in the head kidney,skin,liver and other tissues of female discus,and significantly high expression was found in the head kidney and skin.The results of in situ hybridization found that the mrc gene was expressed in the epidermal mucus layer of the skin of discus.In conclusion,under the conditions of this experiment,we conducted a preliminary exploration on the regulation mechanism of genes related to "milk " secretion in parental care behavior of discus,and provided basic information for the regulation mechanism of parental care of discus. |
PDF Full Text Request