翘嘴鲌Runx2b基因的克隆与表达特征分析  

Cloning and expression analysis of Runx2b in Culter alburnus

作　　者：何昌熙 郑建波 马建波 贾永义 刘士力[2] 蒋文枰[2] 迟美丽[2] 程顺[2] 李飞[2] HE Changxi;ZHENG Jianbo;MA Jianbo;JIA Yongyi;LIU Shili;JIANG Wenping;CHI Meili;CHENG Shun;LI Fei(Key Laboratory of Freshwater Aquatic Genetic Resources,Ministry of Agriculture and Rural Affairs,College of Fisheries and Life Science,Shanghai Ocean University,Shanghai 201306,China;Key Laboratory of Freshwater Aquaculture Genetic and Breeding,Zhejiang Institute of Freshwater Fisheries,Huzhou 313001,Zhejiang,China;Yuyao Aquaculture Technology Extension Center,Yuyao 315400,Zhejiang,China)

机构地区：[1]上海海洋大学水产与生命学院,农业农村部淡水水产种质资源重点实验室,上海201306 [2]浙江省淡水水产研究所浙江省淡水水产遗传育种重点实验室,浙江湖州313001 [3]余姚市水产技术推广中心,浙江余姚315400

出　　处：《浙江农业学报》2024年第5期1024-1031,共8页Acta Agriculturae Zhejiangensis

基　　金：浙江省农业(水产)新品种选育重大科技专项(2021C02069-3);浙江省自然科学基金(LTGN23C190008)。

摘　　要：为探究Runx2b基因在翘嘴鲌肌间刺骨化过程中的调控作用,基于转录组数据和PCR扩增技术克隆获得了翘嘴鲌Runx2b基因全长编码序列。通过茜素红整体骨骼染色对肌间刺(IBs)形成的不同阶段进行了分期,同时采用qRT-PCR技术检测了Runx2b基因的时空表达水平。结果显示,Runx2b基因开放阅读框为1 401 bp,编码466个氨基酸,预测分子量为50.676 ku,理论等电点为9.35;氨基酸序列多重比对显示,翘嘴鲌Runx2b氨基酸序列与近源物种的同源性高达95%以上,且都具有高度保守的Runt和RunxI结构域;系统进化树分析表明,翘嘴鲌与团头鲂的亲缘关系最为接近。茜素红染色结果显示,在肌间刺发育前,翘嘴鲌的主轴骨骼和附肢骨骼已经发育完全;其中,孵化后15 d,尾部肌隔中开始出现小肌间刺;孵化后30 d,翘嘴鲌幼体的肌间刺发育完全。组织表达结果显示,Runx2b基因在翘嘴鲌各组织中均有表达,鳃中的表达量最高,其次是肌肉。此外,Runx2b基因在翘嘴鲌幼体不同生长阶段的表达模式与肌间刺骨化发生的时序保持一致,提示该基因与肌间刺骨化的相关性。研究结果可以为后续无肌间刺翘嘴鲌新种质的创制和肌间刺形成的分子机制提供基础数据。In order to explore the preliminary function of Runx2b gene of Culter alburnus during the ossification process of intermuscular bones(IBs),we obtained the full-length coding sequence of Runx2b gene by transcriptome data and PCR verification.In this study,we staged the different periods of IBs formation by alizarin red staining,and spatial and temporal expression level of Runx2b were investigated by qRT-PCR.The results showed that the open reading frame(ORF)of Runx2b was 1401 bp,which encoded 466 amino acids with a predicted molecular weight of 50.676 ku and a theoretical isoelectric point of 9.35.The results of amino acid multi-alignment showed that the topmouth culter Runx2b protein displayed a higher identity of 95%with other cyprinidae fish specie,and shared highly conserved Runt and RunxI domains.Phylogenetic tree analysis showed that the topmouth culter Runx2b was most closely related to that of Megalobrama amblycephala.Alizarin red staining showed that the fins and axial skeleton had developed completely before IBs formation.A few IBs of small length emerged in the tail at 15 d,and had a mature morphology and length at 30 d.In addition,mRNA expression abundance was detected in all tested tissues,with the highest expression level in the gill,followed by muscle.Moreover,the expression pattern of Runx2b gene at different growth stages of juveniles was highly consistent with the timing of IBs ossification,indicating its regulation role in IBs development.Our data will contribute to generating IBs-free new varieties and revealing the mechanism of IBs formation.

关 键 词：翘嘴鲌 肌间刺 Runx2b 基因表达 骨化 

分 类 号：S917[农业科学—水产科学]