Cloning And Functional Characterization Of Leptin And Its Receptors In Quail

The authenticity of the “chicken leptin gene” was questioned since it was first reported in 1998.Although leptin receptor(LEPR)gene had been widely confirmed in poultry,its physiological functions were still ambiguous due to the lack of endogenous leptin.In 2014,the successful cloning of bird Leptins have partly answered the question whether there are Leptin gene in avain which it has blocked the research of avian Leptin/LEPR for a long time,and have overcome the research bottlenecks of avian Leptin/LEPR.Since then,the cloning and functionial study of Leptin/LEPR in avian especially in poultry have become a hotspot.On account of blastn in public database and analysis,we firstly cloned the Leptin and LEPR gene of quail,and then analysed their structures and identified that quail Leptin is the orthologous gene.We investigated the pattern of tissue expression by using the real time fluorescent quantitative PCR.To investigate the effects of Leptin/LEPR on regulatory of reproduction,Korea female quails at 3 weeks(juvenile)and at 20 weeks(mature)were taken as objects of study to monitor the expression difference of the reproduction related genes and Leptin/LEPR of gonad axis in the two developmental stage.To investigate the effects of Leptin/LEPR on regulation of food intake,Korea female quails at 3 weeks(juvenile),which were fasted and refed,were taken as objects of study to monitor the expression difference of orexigenic genes,anorexigenic genes and Leptin/LEPR in different treatment states.The results were as follows:1.Based on the published sequences of avian Leptin,a partial exon4 sequence of quail Leptin was obtained by homologous sequence searching and matching in SRA database of Japanese quail and the cloning verification.Sequence analysis indicated that the sequence with 64% GC content.The deduced amino acid sequence of exon4 of quail Leptin shared relatively low identities with Leptins of other vertebrates,but possessed the conserved domains.2.The CDS sequence of quail LEPR was firstly obtained by using the degenerate primer design and the PCR cloning technology.Meanwhile,the four different alternative splicing variants were also identified including one long LEPR variant(q LEPRl)and three alternative splicing variants(q LEPR-a,q LEPR-b and q LEPR-c).Among the isoforms,q LEPRl contained all the motifs,domains,and basic tyrosine residues that are conserved in all other vertebrate LEPRs.While the other three isoforms lacked transmembrane domain,intracellular domain and binding domain and therefore belonged to soluble truncated LEPR proteins.Evolutionary analysis showed that there existed a similar evolutionary relationship between Leptin and LEPR in vertebrate and that there was a high homology Leptin or LEPR between quail and poultry especially galliformes.3.The pattern of tissue expression of quail Leptin/LEPR were explored by using the real time fluorescent quantitative PCR.The results showed that Leptin and LEPR was widely expressed in all examined tissues of quail,which prompt that quail Leptin has diverse biological functions.And there were relatively higherexpression in skeletal muscle,pancreas and hypothalamus of quail but LEPR were extremely high expression in pituitary gland,which prompt that Leptin/LEPR play an important role in brain control of energy balance,reproductive regulation and muscle development of quail.Meanwhile,the expression of Leptin/LEPR were lower level in liver and adipose tissue of quail,which indicate that quail Leptin is not a fat or an adipose secretion of hormone.4.The part made a primary study to the relationship between Leptin/LEPR and regulatory of quail reproduction via exploring the differential expression of reproduction related genes and Leptin/LEPR in different developmental stages.The results showed that the expression of reproduction related genes(Gn RH-I?FSH-??FSHR and LHR)were up-regulated in the gonad axis of mature quails,compared to that in 3-week quails.And the gonad axis of mature quail was activated.Meanwhile,the expression of Leptin and t-q LEPR(including all the isoforms)were also un-reglated in hypothalamic and pituitary of mature quails,which indicated that there may exist a certain positive correlation between Leptin/LEPR and the activation of reproductive axis.Along with quail sexual maturity and entering the peak of egg production,follicle stimulating development and ovarian follicle stimulating hormone secretion of genes(LHR and FSHR)were up-regluated expression,while The expression of Leptin/t-q LEPR and the apoptosis gene(caspase3)were down-regulated in ovary of mature quails,which indicated that there may exist a certain negative correlation between Leptin/LEPR and quail follicle development.5.The part made a primary study to the relationship between Leptin/LEPR and regulation of food intake via exploring the differential expression of orexigenic genes(Ag RP/NPY),anorexigenic genes(POMC and MC4R)and Leptin/LEPR in 3-week fasted and refed quails.The results showed that the expression of Ag RP/NPY were significantly up-regulated in fasting of quail,but were significantly down-regulated in refeeding of quail.The expression of POMC and MC4 R were down-regulated in different degrees in the two different physiological status.However,the expression of Leptin/LEPR were up-regulated in fasting,and down-regulated in refeeding,which indicated that Leptin may be not an anorexia factor in regulation of quail food intake.In conclusion,quail Leptin and its receptor LEPR were cloned for the first time.And they possess the unique tissue expression pattern which are different to the homologue of other species.quail Leptin was not a fat or an adipose secretion of hormone,whichplay an important role in brain control of energy balance,reproductive regulation and muscle development.There may exist a certain positive correlation between Leptin/LEPR and the activation of reproductive axis,while may exist a certain negative correlation between Leptin/LEPR and quail follicle development.And Leptin may be not an anorexia factor in regulation of quail food intake.