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

Abstract

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

CLC Number:

S917.4

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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Cloning,Sequence Analysis and Spationtemporal Expression of Runx2b Gene in Qihe Crucian Carp (Carassius auratus)

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