Tissue Distribution And Expression Differences At Different Temperatures Of GHRH And GHRHR In Trachinotus Blochii

Growth hormone releasing hormone(GHRH)is a protein hormone,the main biological function of which is to stimulate the secretion of growth hormone in the pituitary cells.It plays an important role in the growth,development and metabolism of vertebrates by binding to receptor of GHRH.The Trachinotus blochii is a rapid growth of marine fish.In order to reveal the mechanism of metabolism and feeding,this paper studied the expression patterns of GHRH and GHRHR genes in different temperatures and preprandial postprandial.The full-length cDNA sequence of GHRH gene was cloned in T.blochii by RACE technology.The results showed that the full length of GHRH cDNA was 1019 bp,including a 5'untranslated regions(UTR)of 327 bp and a 3'UTR of 164 bp.The open reading frame(ORF)was 528 bp,which encoded a putative protein of 175 amino acids.The results of homology analysis showed the homology between GHRH gene of of T.blochii with the same Perciformes of Epinephelus coioides were 97.1%,and the relationship was closer;The full-length of TbGHRHR gene was of 1141 bp,including a 5'untranslated regions(UTR)of 66 bp and a 3'UTR of 469 bp.The open reading frame(ORF)was 606 bp,which encoded a putative protein of 201 amino acids.The results of homology analysis showed the homology between GHRHR gene with other perciformes fish was more than 86%.The phylogenetic analysis showed that the GHRHR gene of T.blochii and S.dumerili clustered into a branch,and the relationship was closer.The expression of Tb GHRH and GHRHR gene in different tissues and embryonic period by real-time fluorescence quantitative PCR.The results of tissue expression show that the expression region of Tb GHRH gene were mostly in the central nervous system,in which the hypothalamus expression was the highest,and other peripheral tissues have hardly been detected.The Tb GHRHR transcripts were detectable in the telencephalon,mesencephalon,cerebellum,hypothalamus,pituitary and other brain regions.Its highest expression was observed in the pituitary.In addition to a small amount of heart,intestinal and gonadal expression in other tissues.It was not detected in the rest of the tissues.Expression results of various embryonic stages show that The Tb GHRH and GHRHR was detected during fertilization and subsequent development,and the expression level reached the highest in larval stage.At different temperatures(21 ?;25 ?;29 ?;33 ?),the results of temperature control experiments show the expression of GHRH mRNA in hypothalamus of T.blochii was significantly different under different temperature gradients,of which the expression level of GHRH mRNA was the highest at 21 ? in the low temperature group(P<0.05),and the expression level of GHRH mRNA in the temperature group at 25 ? was also higher than that of the temperature group at 29 ? and at 33 ?,But it is no significant difference(P>0.05);the expression of GHRHR mRNA in pituitary of T.blochii was also significantly different under different temperature gradients,of which the expression level of GHRHR mRNA was the highest at 29 ?(P<0.05),and the expression level of GHRH mRNA in the temperature group at 25 ? was also higher than that of the low temperature group at 21 ? and high temperature group at 33 ?(P<0.05).Periprandial(pre-and post-feeding)changes of GHRH and GHRHR mRNA expressions in the hypothalamus and pituitary were examined.Seven groups of weight-matched T.blochii(average body weight 68.01±4.34g,n=10/group)were acclimated for one week to tank conditions.Hypothalamus and pituitary were collected at:3 h prior to feeding(04:00 h),1 h prior to feeding(06:00 h),upon commencement of feeding(07:00 h),1 h after feeding(08:00 h)and 3 h after feeding(10:00 h).Two unfed groups were sampled at 1 h and 3 h and served as the fasted group.The results of postprandial experiments show the expression of GHRH mRNA in hypothalamus and GHRHR mRNA in pituitary of T.Blochii were significantly increased at 1 h after meal(P<0.05),but significantly decreased at 3 h after meal(P<0.05).The research can draw the following conclusions,the present study first cloned the full-length GHRH and GHRHR cDNA from the T.Blochii.Besides,the expression region of Tb GHRH and GHRHR gene were mostly in the brain tissue.Furthermore,the expression of GHRH and GHRHR genes were also affected in different temperature environments and feeding levels,the GHRH and GHRHR are negatively correlated with temperature,and these two genes may have potential role in feeding regulation of T.Blochii.The results of this study can provide an essential groundwork to further elucidate the growth-metabolism and appetite regulatory systems of GHRH and GHRHR in teleosts.