淇河鲫Runx2b基因克隆、序列分析及时空表达

doi:10.16431/j.cnki.1671-7236.2025.06.024

摘要/Abstract

摘要： 【目的】成骨分化特异转录因子2b(Runx2b)基因在调节鱼类骨骼发育方面具有重要作用。本研究旨在探究淇河鲫(Qihe crucian carp,Carassius auratus)Runx2b基因的特征和表达情况。【方法】参考银鲫Runx2b基因序列(GeneBank登录号：NC_068415.1)结合本地基因组数据进行BLAST比对设计引物，通过PCR分段克隆Runx2b基因，并分别通过DNAStar和Mega 11.0软件进行序列相似性比对及系统发育树构建。通过ORF finder和ProtParam等在线网站进行生物信息学分析。利用实时荧光定量PCR技术检测Runx2b基因在淇河鲫不同发育时期和不同组织中的表达水平。【结果】测序拼接比对后得到Runx2b-A和Runx2b-B基因，其CDS区均为1 392 bp，编码463个氨基酸。两基因的CDS序列相似性为95.33%，Runx2b-A和Runx2b-B氨基酸序列均与银鲫对应的氨基酸序列相似性最高，分别为98.0%和99.1%。系统进化树分析显示，淇河鲫与银鲫亲缘关系最近。生物信息学分析表明，两个Runx2b蛋白分子式分别为C₂₂₀₆H₃₄₀₉N₆₃₇O₆₈₅S₁₈和C₂₁₉₆H₃₃₉₅N₆₃₃O₆₉₅S₁₆，均属于亲水性不稳定蛋白，且蛋白质结构主要以无规则卷曲为主，两个蛋白质序列中都存在Runx家族蛋白特征性的Runt-dom和RunxⅠ保守结构域。实时荧光定量PCR检测结果表明，Runx2b-A和Runx2b-B基因在淇河鲫整个肌间刺发育阶段整体表达趋势较为一致，肌间刺发育前期(1～5 dph)表达量较高，在肌间刺开始发育后(5～13 dph)表达量逐渐降低，在后期表达量出现上升趋势，尤其在肌间刺发育晚期(25～45 dph)仍有较高的表达量；Runx2b-A和Runx2b-B基因在淇河鲫脑、肝脏、背部肌肉、尾部肌肉等9个组织中均有表达，二者均在脑和肝脏中表达量较高，且显著高于其他组织(P＜0.05)，二者在尾部肌肉中的相对表达量均显著高于背部(P＜0.05)，但在不同组织中呈现差异表达，具有不同的表达水平。【结论】本研究成功克隆获得两条淇河鲫同源基因Runx2b-A和Runx2b-B的CDS序列，二者氨基酸序列与鲤科鱼类的相似较高，蛋白质序列中存在Runx家族蛋白特征性的Runt-dom和RunxⅠ保守结构域。淇河鲫两个Runx2b基因表达具有广泛的组织分布，但在不同组织中呈现差异表达，具有不同的表达模式；在不同发育时期整体表达趋势较为一致，在肌间刺发育晚期仍有较高的表达量，可为后续深入研究肌间刺发育机制和创制无肌间刺淇河鲫奠定基础。

关键词: 淇河鲫; Runx2b基因; 克隆; 序列分析; 时空表达

Abstract: 【Objective】 The osteogenic differentiation-specific transcription factor 2b (Runx2b) gene played an important role in regulating fish bone development.This study aimed to explore the characteristics and expression of Runx2b gene in Qihe crucian carp (Carassius auratus).【Method】 Primers were designed by referring to the Runx2b gene sequence of Carassius gibelio (GeneBank accession No.:NC_068415.1) and conducting BLAST alignment with local genomic data.The Runx2b gene was segmented and cloned using PCR technology,and sequence similarity alignment and phylogenetic tree construction were carried out using DNAStar and Mega 11.0 software, respectively.Online websites such as ORF finder and ProtParam were used for bioinformatics analysis.At the same time, Real-time quantitative PCR was used to detect the expression of Runx2b gene in different developmental stages and different tissues of Qihe crucian carp.【Result】 After sequencing and alignment,Runx2b-A and Runx2b-B genes were obtained,with CDS regions of 1 392 bp each,encoding 463 amino acids.The similarity of the CDS sequences of the two genes was 95.33%.The amino acid sequence of Runx2b-A had the highest similarity with that of Carassius gibelio, at 98.0% and 99.1%,respectively. Phylogenetic tree analysis showed that Qihe crucian carp was most closely related to Carassius gibelio.Bioinformatics analysis showed that the molecular formulas of the two Runx2b proteins were C₂₂₀₆H₃₄₀₉N₆₃₇O₆₈₅S₁₈ and C₂₁₉₆H₃₃₉₅N₆₃₃O₆₉₅S₁₆,respectively.Both belong to hydrophilic and unstable proteins,and the protein structure was mainly composed of random coils.The characteristic Runt-dom and RunxⅠ conserved domains of Runx family proteins exist in both protein sequences.The results of Real-time quantitative PCR showed that the overall expression trends of Runx2b-A and Runx2b-B genes were relatively consistent during the whole development stage of intermuscular bones in Qihe crucian carp.The expression were relatively high in the early stage of intermuscular bone development (1-5 dph).After the intermuscular bones started to develop (5-13 dph),the expression of Runx2b genes were gradually decrease,and then showed an upward trend in the later stage,especially in the late stage of intermuscular bone development (25-45 dph),where the expression were still relatively high.The Runx2b-A and Runx2b-B genes were expressed in 9 tissues such as the brain,liver,back muscle,and tail muscle of Qihe crucian carp.Both genes had relatively high expression levels in brain and liver,which were significantly higher than those in other tissues (P＜0.05).The relative expression of both genes in tail muscle were significantly higher than those in back muscle (P＜0.05),but they showed differential expression in different tissues with different expression.【Conclusion】 In this study,the CDS sequences of two homologous genes,Runx2b-A and Runx2b-B,of Qihe crucian carp were successfully cloned.The amino acid sequences of the two genes had relatively high similarity with those of fish in Cyprinidae.The characteristic Runt-dom and RunxⅠ conserved domains of Runx family proteins existed in the protein sequences.The two Runx2b genes in Qihe crucian carp were widely distributed in tissues but show differential expression in different tissues with different expression patterns.The overall expression trends were relatively consistent in different developmental stages,and the expression were still relatively high in the late stage of intermuscular bone development.This study coould lay a foundation for further research on the development mechanism of intermuscular bones and the creation of Qihe crucian carp without intermuscular bones.

Key words: Qihe crucian carp; Runx2b gene; cloning; sequence analysis; spationtemporal expression

中图分类号:

S917.4

闪金研, 连凯琪, 马峻, 刘玉玲, 聂竹兰, 李斌顺, 彭仁海, 魏杰. 淇河鲫Runx2b基因克隆、序列分析及时空表达[J]. 中国畜牧兽医, 2025, 52(6): 2717-2728.

SHAN Jinyan, LIAN Kaiqi, MA Jun, LIU Yuling, NIE Zhulan, LI Binshun, PENG Renhai, WEI Jie. Cloning,Sequence Analysis and Spationtemporal Expression of Runx2b Gene in Qihe Crucian Carp (Carassius auratus)[J]. China Animal Husbandry & Veterinary Medicine, 2025, 52(6): 2717-2728.

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