多浪羊CNP基因克隆及其在初情期组织表达特性分析

doi:10.16431/j.cnki.1671-7236.2022.04.022

Abstract

Abstract: 【Objective】 The C-type natriuretic peptide (CNP) gene of Dolang sheep was cloned and analyzed,and its expression pattern in hypothalamus,pituitary gland,oviduct,ovary and uterus before and after the onset of puberty in Dolang sheep was explored,in order to provide a reference for studying the mechanism of CNP gene in the onset of puberty.【Method】 Primers were designed with reference to the sequence of sheep CNP gene in GeneBank (accession No.:XM_027974523.1),and RT-PCR technique was used to amplify CNP gene of Dolang sheep before,during and after the puberty,and cloning and sequencing were performed.DNAMAN software was used to compare similarity with other species and Mega 5.0 was used to construct phylogenetic tree.The nucleotide sequence of CNP gene and the physicochemical properties such as hydrophobicity,signal peptide,phosphorylation site,transmembrane domain,secondary and tertiary structures of the encoded proteins were analyzed using bioinformatics softwares.The expression of CNP gene in hypothalamus,pituitary,oviduct,ovary and uterus of Dolang sheep was examined using Real-time quantitative PCR technique.【Result】 The sequence size of the CNP gene of Dolang sheep was 2 227 bp,including 5'-UTR 50 bp,3'-UTR 914 bp and CDS region 1 263 bp.Similarity comparison and phylogenetic tree results showed that Dolang sheep had the closest affinity to goat and the most distant affinity to chicken.The CNP gene of Dolang sheep encoded a total of 420 amino acids and its encoding protein was a hydrophilic protein with no transmembrane structural domain or signal peptide.The secondary structure of the CNP protein in Dolang sheep was mainly α-helix and random coil.The tertiary structure prediction showed that the CNP protein had no ligand structure.Real-time quantitative PCR results showed that the CNP gene was significantly more expressed in the hypothalamus than in other tissues at pre-puberty,puberty and post-puberty (P<0.05),CNP gene expression in the hypothalamus was significantly lower in the puberty than in the pre-puberty phase (P<0.05).In uterus,the expression level of CNP gene was significantly higher in pre-puberty than puberty and post-puberty (P<0.05).【Conclusion】 The CNP gene was successfully cloned in Dolang sheep,which was mainly expressed in hypothalamus and uterus.The expression of CNP gene in hypothalamus tissue decreased and then increased during the developmental process pre-puberty,puberty and post-puberty suggesting that CNP gene might be involved in the regulation of the initiation process during pubertal phase.

Key words: Dolang sheep; C-type natriuretic peptide; gene cloning

CLC Number:

S826.8

ZHANG Zhishuai, SUI Zhiyuan, ZHANG Jihu, LI Qingjin, XING Feng. Cloning of CNP Gene and Its Tissue Expression Characteristics at Puberty in Dolang Sheep[J]. China Animal Husbandry and Veterinary Medicine, 2022, 49(4): 1402-1412.

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Cloning of CNP Gene and Its Tissue Expression Characteristics at Puberty in Dolang Sheep

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