半番鸭NMB基因克隆、生物信息学和组织表达分析

doi:10.16431/j.cnki.1671-7236.2025.04.023

摘要/Abstract

摘要： 【目的】本研究旨在克隆半番鸭神经介素B (NMB)基因并对其进行序列分析，检测NMB基因在半番鸭不同组织中的表达情况。【方法】根据NCBI数据库中鸭NMB基因的预测序列(登录号：XM_027466185.2)设计引物，通过PCR扩增获得半番鸭NMB基因片段，克隆后测序获得NMB基因序列；利用生物信息学分析软件对半番鸭NMB核苷酸和氨基酸序列进行分析，并构建遗传进化树；通过实时荧光定量PCR检测NMB基因mRNA在半番鸭体内多个组织中的表达情况。【结果】本试验克隆获得了半番鸭NMB基因，其CDS序列长度为387 bp，编码128个氨基酸，包括14个氨基酸(GNLWATGHFMGKKS)的蛙皮素保守结构域，与鸡、小鼠、猪、牛和人的NMB的保守结构域完全相同。相似性比对发现，半番鸭与鸡NMB基因核苷酸和氨基酸序列的相似性较高，分别为87.9%和89.8%，与其他动物的相似性较低；半番鸭NMB在进化上与鸡亲缘关系较接近，与牛、马、猪、犬等家畜较远。生物信息学分析结果表明，半番鸭NMB蛋白属于亲水性蛋白，其N-端22个氨基酸为信号肽，二级结构和三级结构中α-螺旋、延伸链、β-转角和无规则卷曲分别占53.91%、14.06%、7.03%和25.00%，半番鸭NMB蛋白主要与NMBR和GRPR蛋白结合发挥作用。实时荧光定量PCR结果显示，NMB基因mRNA在半番鸭中枢和外周多个组织器官中均有表达；以空肠为对照，NMB基因在中枢的小脑、大脑和视叶表达量相对较高(P＜0.01)，在外周的气管、肺脏、松果体、甲状腺、肾脏和尾脂腺表达量相对较高(P＜0.01)。另外，NMB基因mRNA在不同性别半番鸭体内的表达趋势基本一致，但在在小脑和胰腺等组织中母鸭比公鸭NMB基因mRNA的表达量相对较高(P＜0.01)，但在松果体和肾脏等组织中则相反(P＜0.01；P＜0.05)。【结论】半番鸭NMB蛋白具有蛙皮素的保守结构域，与鸡的亲缘关系最近、蛋白相似性高，属于亲水性蛋白，有信号肽。NMB基因在半番鸭多个中枢(小脑、大脑和视叶)和外周(松果体、肾脏、尾脂腺、甲状腺和气管等)组织器官中高表达。以上结果可为进一步研究半番鸭NMB基因的功能提供参考。

关键词: 半番鸭; NMB基因; 克隆; 表达; 生物信息学

Abstract: 【Objective】 The purpose of this study was to clone and sequence the neuromedin B (NMB) gene from Mulard duck,and to detect the expression of NMB gene in Mulard duck.【Method】 According to the predicted NMB gene sequence of duck in the NCBI database (accession No.：XM_027466185.2),primers were designed to amplify NMB gene fragment in Mulard duck by PCR amplification,and the NMB gene sequence was obtained by cloning and sequencing.The nucleotide and amino acid sequences of NMB in Mulard duck were analyzed using bioinformation analysis software,and the phylogenetic tree was constructed.Real-time quantitative PCR was used to detect the expression of NMB gene mRNA in multiple tissues of Mulard duck.【Result】 In this study,NMB gene of Mulard duck was cloned with 387 bp CDS sequence and encoding 121 amino acids,including 14 amino acids (GNLWATGHFMGKKS) of the conserved domain of bombesin,which was identical to that in NMB of Gallus gallus，Mus musculus，Sus crofa，Bos taurus and Homo sapiens.The similarity analysis showed that the similarity of nucleotide and amino acid sequences of NMB between Mulard duck and Gallus gallus were 87.9% and 89.8%,respectively,and the similarity with other animals were lower.In addition,NMB of Mulard duck was evolutionarily close to Gallus gallus,and far from domestic animals such as Bos taurus, Equus caballus,Sus crofa and Canis lupus familiaris.The results of bioinformatics analysis showed that NMB protein of Mulard duck was a hydrophilic protein,and its N-terminal 22 amino acids were signal peptides.In the secondary and tertiary structures,the proportion of alpha helix,extended chain,beta turn and random coil were 53.91%,14.06%,7.03% and 25.00%,respectively.The NMB protein of Mulard duck mainly binded to NMBR and GRPR proteins.Real-time quantitative PCR results showed that NMB gene mRNA was expressed in the central and peripheral tissues and organs of Mulard duck.Compared with the jejunum,the expression levels of NMB gene were relatively high in the central cerebellum,brain and optic lobe (P＜0.01),and relatively high in the peripheral trachea,lung,pineal gland,thyroid,kidney and uropygial gland (P＜0.01).In addition,the expression trend of NMB gene mRNA in Mulard duck of different genders was basically the same,but there were different expression in female and male ducks in individual tissues.The expression of NMB gene mRNA in female duck was relatively higher than that in male duck in cerebellum and pancreas (P＜0.01),but the opposite was true in pineal gland and kidney (P＜0.01 or P＜0.05).【Conclusion】 NMB protein in Mulard duck had the conserved domain of bombesin.The genetic relationship was the closest between Mulard duck and Gallus gallus,and the protein similarity was high.NMB protein was hydrophilic protein and had signal peptide.NMB gene was highly expressed in the central (cerebellum,brain and optic lobe) and peripheral (pineal gland,kidney,uropygial gland,thyroid gland and trachea,etc.) tissues and organs of Mulard duck.The results could provide reference for further study on the function of NMB gene in Mulard duck.

Key words: Mulard duck; NMB gene; cloning; expression; bioinformatics

中图分类号:

S834⁺.89

张莹, 陈广振, 刘梅, 李红霞, 俞燕, 贾雪波, 李雯倩, 范建华, 马志禹. 半番鸭NMB基因克隆、生物信息学和组织表达分析[J]. 中国畜牧兽医, 2025, 52(4): 1705-1715.

ZHANG Ying, CHEN Guangzhen, LIU Mei, LI Hongxia, YU Yan, JIA Xuebo, LI Wenqian, FAN Jianhua, MA Zhiyu. Cloning,Bioinformatics and Tissue Expression Analysis of NMB Gene in Mulard Duck[J]. China Animal Husbandry and Veterinary Medicine, 2025, 52(4): 1705-1715.

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