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

摘要/Abstract

摘要： 【目的】克隆多浪羊C型利钠肽(CNP)基因,并进行生物信息学分析,探讨其在多浪羊初情期启动前后下丘脑、垂体、输卵管、卵巢、子宫中的表达规律,以期为研究CNP基因在多浪羊初情期启动过程中的作用机制提供参考。【方法】参考GeneBank中绵羊CNP基因的序列(登录号:XM_027974523.1)设计引物,以初情期前、初情期、初情期后的多浪羊为试验对象,采用RT-PCR技术扩增多浪羊CNP基因并进行克隆测序;用DNAMAN软件同其他物种进行相似性比对,并使用Mega 5.0构建系统发育树。用生物信息学软件分析CNP基因的核苷酸序列及其编码蛋白的疏水性、信号肽、磷酸化位点、跨膜结构域等理化性质和二级结构、三级结构信息;用实时荧光定量PCR技术检测多浪羊下丘脑、垂体、输卵管、卵巢、子宫中CNP基因的表达量。【结果】多浪羊CNP基因序列大小为2 227 bp,其中包括5'-UTR 50 bp、3'-UTR 914 bp和CDS区1 263 bp。相似性比对和系统发育树结果显示,多浪羊与山羊的亲缘关系最近,与鸡的亲缘关系最远;多浪羊CNP基因共编码420个氨基酸,其编码蛋白是亲水性蛋白质,无跨膜结构域和信号肽。多浪羊CNP蛋白的二级结构主要是α-螺旋和无规则卷曲;三级结构预测显示CNP蛋白无配体结构;实时荧光定量PCR结果显示,在初情期启动的3个发育阶段,下丘脑中CNP基因表达量显著高于其他组织(P<0.05);初情期下丘脑中CNP基因的表达量显著低于初情期前(P<0.05);子宫中初情期前CNP基因的表达量显著高于初情期和初情期后(P<0.05)。【结论】本研究成功克隆了多浪羊CNP基因,其主要在下丘脑和子宫中表达;在初情期前、初情期、初情期后的发育过程中,下丘脑组织中CNP基因的表达量先降低再升高,提示CNP基因可能在初情期的启动过程中参与调控。

关键词: 多浪羊; C型利钠肽; 基因克隆

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

中图分类号:

S826.8

张志帅, 隋志远, 张继虎, 李庆瑾, 邢凤. 多浪羊CNP基因克隆及其在初情期组织表达特性分析[J]. 中国畜牧兽医, 2022, 49(4): 1402-1412.

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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