多浪羊NPTX1基因克隆、生物信息学分析及在初情期的表达研究

doi:10.16431/j.cnki.1671-7236.2023.03.020

摘要/Abstract

摘要： 【目的】试验旨在克隆多浪羊神经元正五聚体蛋白1(neuronal pentraxin 1,NPTX1)基因CDS序列,利用生物信息学分析NPTX1蛋白的结构和功能,并研究其在多浪羊初情期启动过程中性腺轴不同组织中的表达规律。【方法】采集初情期前、初情期、初情期后健康雌性多浪羊的下丘脑、垂体、输卵管、卵巢、子宫组织,提取总RNA并反转录成cDNA,以初情期前下丘脑cDNA为模板,PCR扩增多浪羊NPTX1基因并克隆测序,对其进行生物信息学分析;采用实时荧光定量PCR分析NPTX1基因在多浪羊下丘脑、垂体、卵巢、子宫、输卵管5个组织中的表达水平。【结果】克隆得到NPTX1基因长1576 bp,其中CDS区序列为1299 bp,与GenBank上预测的绵羊mRNA序列(登录号:XM_005683183.3)相似性达99.85%。系统进化树分析得出,多浪羊NPTX1基因与绵羊的遗传距离最近,与鸡的遗传距离最远。生物信息学分析发现,NPTX1蛋白为不稳定的亲水性酸性蛋白,具有1个信号肽,属于分泌蛋白;二级结构中α-螺旋和无规则卷曲占比分别为41.90%和40.73%,与三级结构预测结果一致。实时荧光定量PCR结果显示,NPTX1基因在多浪羊3个时期5个不同性腺组织中均有表达,在初情期下丘脑中表达量最高,显著高于初情期前和初情期后(P＜0.05)。【结论】试验成功克隆了多浪羊NPTX1基因CDS全长序列,并揭示了NPTX1基因在多浪羊初情期不同阶段性腺轴组织中的表达差异性,为进一步探究NPTX1基因在多浪羊初情期启动过程中的调控机制提供依据。

关键词: 多浪羊; NPTX1基因; 克隆; 初情期; 组织表达

Abstract: 【Objective】 This study was aimed to clone the coding region (CDS) sequence of neuronal pentraxin 1 (NPTX1) gene in Dolang sheep,analyze its structure and function by bioinformatics,and study its expression pattern in different tissues of gonadal axis during puberty initiation in Dolang sheep.【Method】 The hypothalamus,pituitary,oviduct,ovary and uterus tissues of healthy female Dolang sheep were collected from prepubertal,pubertal and postpubertal.The total RNA was extracted and reverse transcribed into cDNA.NPTX1 gene of Dolang sheep was amplified by PCR using prepubertal hypothalamus cDNA as template,cloned and sequenced,and its bioinformatics analysis was carried out.The expression of NPTX1 gene in hypothalamus,pituitary,ovary,uterus and oviduct of Dolang sheep were analyzed by Real-time quantitative PCR.【Result】 The cloned NPTX1 gene was 1 576 bp,of which the CDS sequence was 1 299 bp,and the similarity with the mRNA sequence of sheep predicted on GenBank(accession No.:XM_005683183.3) was 99.85%.Phylogenetic tree analysis results showed that NPTX1 gene of Dolang sheep had the closest genetic distance to Ovis aries and the farthest genetic distance to Gallus gallus.Bioinformatics analysis results showed that NPTX1 protein was an unstable hydrophilic acidic protein with a signal peptide and belonged to secretory protein.In the secondary structure of NPTX1 protein,alpha helix and random coil accounted for 41.90% and 40.73%,respectively,which were consistent with the predicted results of the tertiary structure.Real-time quantitative PCR results showed that NPTX1 gene was expressed in five different gonad tissues of Dolang sheep in three stages,and the expression was the highest in hypothalamus of puberty,which was significantly higher than that of prepuberty and postpuberty (P＜0.05).【Conclusion】 The experiment successfully cloned the full-length CDS sequence of NPTX1 gene in Dolang sheep,and revealed the differences in the expression of NPTX1 gene in gonadal axis tissue of Dolang sheep at different stages of puberty,which provided a basis for further exploring the regulation mechanism of NPTX1 gene in puberty onset of Dolang sheep.

Key words: Dolang sheep; NPTX1 gene; cloning; puberty; tissue expression

中图分类号:

S813.3

张永杰, 隋志远, 张志帅, 王晨光, 李孝君, 邢凤. 多浪羊NPTX1基因克隆、生物信息学分析及在初情期的表达研究[J]. 中国畜牧兽医, 2023, 50(3): 1048-1058.

ZHANG Yongjie, SUI Zhiyuan, ZHANG Zhishuai, WANG Chenguang, LI Xiaojun, XING Feng. Cloning and Bioinformatics Analysis of NPTX1 Gene and Its Tissues Expression at Puberty in Dolang Sheep[J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(3): 1048-1058.

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