| In this study,Cynoglossus semilaevis,a dominant farmed flounder species in China,was used as the research object.Two retinoic acid receptors RAR? and RAR? were cloned and the gene structure were analyzed.The expression patterns of these two RAR genes in various tissues were determined which indicated that they are involved into blind side hypermelanosis.The temporal and spatial expression patterns of these two RAR genes were investigated,and their responses to light intensity were clarified.Furthermore,the relationship between RAR genes expression levels and other body coloration genes expression levels was clarified.Results from the present study would provide more basic knowledge of mechanisms underlying blind side hypermelanosis of tongue sole.1.Cloning and spatial expression characteristics of RAR? and RAR? genes in Cynoglossus semilaevisRT-PCR and RACE methods were used to obtain the cDNA full length sequences of 2 kinds of retinoic acid receptors(C.semilaevis),RAR? and RAR?,and their tissue expression characteristics were analyzed by qPCR technology.The RAR? cDNA sequence of the C.semilaevis is 1823 bp in length,encoding 443 amino acids,and the RAR? cDNA sequence is 1959 bp,encoding 489 amino acids.Homology analysis showed that the homology between RAR? and RAR? was 60.8%,and had strong phylogenetic conservation.Phylogenetic analysis showed that C.semilaevis RAR? and RAR? respectively belong to a separate branch with other fish aggregates.Tissue expression analysis showed that the expression of RAR? mRNA in the kidney was the highest,while the expression of RAR? mRNA in the spleen was the highest.RAR? and RAR? mRNA were detected in other tissues,indicating that the two kinds of RAR of the C.semilaevis may be involved in a variety of physiological processes.The expression of two RAR mRNA in C.semilaevis in ocular skin,blind side blackened skin and blind normal skin increased in turn,and the expression of RAR?in normal eye side skin was higher than that of RAR?,while RAR gamma expression in blind normal skin was significantly higher than that of RAR?,and the expression of RAR? in blind blackened skin was higher than that of RAR?.The differential expression of RAR gene in the ocular side and the blind side skin tissue may be related to the RA/RAR system's regulation of body coloration.2.Temporal expression patterns of two retinoic acid receptors in embryonic development and postembryonic development stages in C.semilaevisThe temporal expression patterns of RAR? and RAR? mRNAs in in gamete,embryo development and postembryonic development stages were investigated in C.semilaevis,and the relationship between their expression levels and other 4 kinds of body coloration related genes expression levels were also investigated.The RAR? and RAR? genes both have maternal and/or paternal inheritance.During the embryonic development stage,the expression levels of these two genes maintained low before the gastrula stage,but increased significantly at the embryo encircling 1/2 of yolk sac stage and lasts till to the hatching stage.These results showed that both RAR? and RAR? were involved in the regulation course of embryonic development of tongue sole.In the postembryonic development stage,the expression level of RAR? mRNA level was high larvae younger than 30 d,but decreased significantly at 40 d,and maintained low expression level to 180 d.The RAR? mRNA level showed the similar expression trend with RAR? mRNA level,and there was no significant difference in the expression level before 30 d,but it increased significantly at 30d(metamorphosis complete point),and decreased significantly after 40 d to a lower level till to 180 d.Statistical analysis showed that the RAR? mRNA expression level in embryonic development stage was positively correlated with the level of MCH1 mRNA expression level,while the RAR? mRNA expression level was significantly positively correlated with the MCH1 mRNA expression level.At the same time,the expression level of POMCb mRNA was positively correlated with the expression level of RAR? mRNA and the expression level of RAR mRNA.As for their expression in postembryonic development stage,there was a significant positive correlation between the RAR? mRNA expression level and the POMCa mRNA expression level,and the expression level of RAR? mRNA was also positively correlated with the expression level of POMCa mRNA.At the same time,the RAR? and RAR? mRNA expression levels were positively correlated with the POMCb mRNA expression level.In the early life stage,the RAR?and RAR? mRNA levels showed a significant positive correlation,which indicated that they may play regulatory roles in the early life stages in a synergistic way.3.Role of retinoic acid receptor in the blind side hypermelanosis of C.semilaevis induced by lightThe effect of four different light intensity conditions on the blind side body color of the young tongue sole was clarified.It was found that under the condition of strong light(500 lx),the weight gain rate of the experimental fish was higher,but it had higher ratio of blind side hypermelanosis and low survival rate.As forthe medium light intensity(300 lx),the experimental fish had higher survival rate,weight gain rate and low blind side hypermelanosis rate,which indicated that it has great potential application in aquaculture of tongue sole.The response of two kinds of retinoic acid receptors including RAR? and RAR? to different light intensity were detected.The high expression level of brain RAR? mRNA was observed in the strong light group,while the highest expression level of eye RAR? mRNA was observed in the weak light group.The expression level of brain RAR? mRNA in the middle light group(300 lx)and the dark group(0 lx)was higher than that in the dark group,while the expression level of eye RAR? mRNA in the middle light group and the weak light group(100 lx)was higher.The expression level of RAR? mRNA in the experimental fish brain without eye blackening in the strong light group and the middle light group was higher than that in the blind side normal individuals,but the expression trend was opposite to the RAR? mRNA expression in the eyes,and the expression of RAR gamma mRNA also had a similar trend.RAR? mRNA There was a strong negative correlation between the expression level of RAR mRNA and POMCa,POMCb and MCH1,but a strong positive correlation with RAR mRNA expression level.The expression level of RAR ? mRNA was positively correlated with the expression level of POMCb mRNA,but has a strong negative correlation with POMCa,MCH2,MCH1 mRNA expression levels,indicating that these two retinoic acid receptor genes,together with other body color related functional genes,participate in the light-induced early development of blind side hypermelanosis of tongue sole in a synergetic or antagonistic way. |
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