【作者】 李晓晖； 张春燕； 冯翠； 张泽文； 冯楠楠； 沙航； 罗相忠； 邹桂伟； 梁宏伟；

【Author】 Li Xiao-hui;Zhang Chun-Yan;Feng Cui;Zhang Ze-Wen;Feng Nan-Nan;Sha Hang;Luo Xiang-Zhong;Zou Gui-Wei;Liang Hong-Wei;Yangtze River Fisheries Research Institude,Chinese Academy of Fishery Sciences;Laboratory of Zooligical Systematics and Application of Hebei Province,College of Life Sciences,Hebei University;Hubei Hongshan Laboratory,Huazhong Agricultural University;

【机构】 中国水产科学研究院长江水产研究所； 河北省动物系统学与应用实验室,河北大学生命科学学院； 华中农业大学湖北洪山实验室；

【摘要】 骨形态发生蛋白6(BMP-6)是TGF-β超家族的一个成员,因为可以刺激骨和软骨形成发育的能力而闻名。近年来,bmp6参与鱼类肌间骨骼形成的研究引起了广泛关注。肋骨是鱼类重要的骨骼结构,对内脏器官起着保护作用。然而,有关bmp6基因对肋骨发育影响的研究还很有限。鲢鱼是中国四大名鱼之一,因其价格低廉、肌肉细嫩而备受青睐。然而,鲢鱼肌间骨骼较多,严重影响了其适口性和加工性能的提高。本研究展示了如何有效利用CRISPR/Cas9技术破坏鲢鱼的bmp6基因,从而在P0代创造出嵌合体,这是首次取得此类成果。嵌合体完全存活,外观正常,部分肌肉间骨骼缺失,其中约30%的嵌合体肋骨分叉或弯曲。随后,对P0嵌合体和野生型的肋骨进行了转录组分析,结果发现有1333个基因表现出差异表达,其中649个基因上调,684个基因下调。KEGG分析结果显示,"NF-kappa B信号通路"、"Hippo信号通路"、"破骨细胞分化"和"造血细胞系"呈现富集,并与骨骼发育显著相关。通路中tnfα、fos和ctgf等基因的上调可能促进破骨细胞的增殖和分化,而通路中tgfb2和tgfbr1等基因的下调可能阻碍成骨细胞的形成和特化,最终导致肋骨异常。本研究就bmp6基因缺失对鲢鱼肋骨发育的影响提出了新的发现,同时探讨了之前尚未探索的鱼类肋骨缺陷的分子机制。更多还原

【Abstract】 Bone morphogenetic protein 6(BMP-6),is a constituent of the TGF-β superfamily,known for its ability to stimulate bone and cartilage formation.The investigation of bmp6’s involvement in the formation of intermuscular bones in fish has garnered significant attention in recent years.The rib cage is an important skeletal structure that plays a protective function for internal organs in fish.However,there has been limited researches on the effects of the bmp6 gene on rib development.Silver carp is one of four major fish in China,favoured for its affordability and tender muscle.Nevertheless,the presence of numerous intermuscular bones in silver carp significantly hinders the advancement of its palatability and suitability for processing.This study showcases the effective utilization of CRISPR/Cas9technology for the purpose of disrupting the bmp6 gene in silver carp,leading to the creation of a chimera in the P0 generation,marking the first instance of such an achievement.The chimeras exhibited complete viability,normal appearance,and partial intermuscular bones loss,with approximately 30% of them displaying rib bifurcation or bending.Subsequently,transcriptome analysis on ribs of P0 chimeras and wild-type was conducted,leading to the identification of 1333 genes exhibiting differential expression,of which 649 were found to be up-regulated and 684 were found to be down-regulated.The results of the KEGG analysis revealed that "NF-kappa B signalling pathway","Hippo signalling pathway","Osteoclast differentiation" and "Hematopoietic cell lineage" exhibited enrichment and displayed a significant correlation with bone development.The up-regulated genes such as tnfα,fos and ctgf in pathways may facilitates the proliferation and differentiation of osteoclasts,whereas the down-regulated of genes such as tgfb2 and tgfbr1 in pathways may hinders the formation and specialization of osteoblasts,ultimately resulting in rib abnormalities.This study presents novel findings on the impact of bmp6 gene deletion on the rib development of silver carp,while simultaneously investigating the previously unexplored molecular mechanisms underlying rib defects in fish.更多还原