Cloning,Promoter Analysis,Tissue Expression And Function Exploration Of Chicken Neurokinin B

NKB belongs to the tachykinin family and is highly conserved among vertebrates.It can participate in many physiological functions,such as regulating anxiety,smooth muscle contraction,and relaxing blood vessels.In recent years,NKB has attracted people’s attention because of its important regulation effect on the reproductive system.At present,there is a lack of relevant research in birds.Chicken is not only a bird model animal,but also an important economic animal.Therefore,in this study,we cloned the full length of chicken cTAC3 cDNA,and explored its promoter,tissue distribution and function.The results are as follows:(1)Using RT-PCR and RACE-PCR technology,the full length of TAC3 cDNA sequence of chicken was cloned and its gene structure was confirmed for the first time.cTAC3 is composed of five exons,with a full length of 787 bp.It can encode the precursor protein containing 132 Aa.The mature peptide(10 Aa)produced by hydrolysis is highly conserved in vertebrates.(2)The cTAC3 promoter region is located in the-1523/+85bp region and contains GATA1,CREB,E2,SP1,GC box,and TATA box.The core promoter is located at-37/+85bp.(3)The qPCR results show that cTAC3 and cTACR3 mRNA have a wide tissue distribution and high expression in the hypothalamus.During chicken development,the expression levels of cTAC3 mRNA in the hypothalamus and pituitary increased,and the expression of cTACR3 mRNA in the hypothalamus increased continuously,while the expression in the pituitary decreased,suggesting that cTAC3/cTACR3 exert potential reproductive regulation in chicken effect.(4)Using pGL3-CRE-luciferase and pGL4-SRE-luciferase reporting systems,it was determined that cNKB can efficiently activate cTACR3 and mediate intracellular cAMP/PKA and MAPK/PKC signaling pathways.The amino acid residue at position 235 of cTACR3 plays an important role in its interaction with cNKB.(5)In vivo test results show that cNKB promotes the expression of GnRH-Ⅰ in the hypothalamus of chicken and stimulates the synthesis of pituitary LH and FSH.Using the chicken pituitary cell culture system,it was confirmed that cNKB can directly act on the pituitary,affect the expression of cFSHβ and cLHβ mRNA.The effect of cNKB on pituitary gland is different in gender,which promotes the expression of female related genes and suppresses male.This study cloned the full length of chicken TAC3 cDNA for the first time,and systematically explored its role and mechanism in chicken.The results not only clarified the function of cNKB in regulating chicken reproduction,but also provided clues for revealing its conservative function in vertebrates.