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中国生物工程杂志

CHINA BIOTECHNOLOGY
中国生物工程杂志
中国生物工程杂志  2021, Vol. 41 Issue (9): 20-26    DOI: 10.13523/j.cb.2105054
研究报告     
斑马鱼hoxa1a基因调控颅面骨骼发育的功能研究*
武秀知1,2,3,王宏杰1,2,3,祖尧1,2,3,**()
1 上海海洋大学水产种质资源发掘与利用教育部重点实验室 上海 201306
2 上海海洋大学科技部海洋生物科学国际联合研究中心 上海 201306
3 上海海洋大学国家水生动物病原库 上海 201306
Functional Study of hoxa1a Regulating Craniofacial Bone Development in Zebrafish
WU Xiu-zhi1,2,3,WANG Hong-jie1,2,3,ZU Yao1,2,3,**()
1 Key Laboratory of Exploration and Utilization of Aquatic Germplasm Resources, Ministry of Education, Shanghai Ocean University, Shanghai 201306, China
2 International Joint Research Center for Marine Biosciences, Shanghai Ocean University, Shanghai 201306, China
3 National Aquatic Animal Pathogen Bank, Shanghai Ocean University, Shanghai 201306, China
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摘要：

hox基因编码一类高度保守的转录因子家族,人类HOXA1的突变会导致阿萨巴斯卡发育不良综合征 (Athabascan brainstem dysgenesis syndrome, ABDS),使人出现因颅骨异常导致的面部畸形和面部麻痹等症状。利用模式生物斑马鱼研究其同源基因hoxa1a的功能机制。首先利用CRISPR/Cas9技术对斑马鱼hoxa1a进行基因编辑,获得了hoxa1a基因突变,T7E1酶切结果显示F0酶切效率平均为70%。F1进一步筛选到两种突变类型,分别是插入了8 bp和删除了7 bp的杂合突变体。杂合子自交得到hoxa1a F2纯合突变体,并且测序验证hoxa1a基因突变成功。5 dpf时,hoxa1a纯合突变体出现颅面发育异常。阿尔新蓝软骨染色和茜素红硬骨染色结果表明,hoxa1a突变体中颅骨发育异常、筛骨板断裂,鳃弓发育出现缺损。成功在斑马鱼中构建ABDS疾病模型,表明hoxa1a突变会造成斑马鱼颅面骨骼发育异常,为其功能机制研究奠定了基础,为人类ABDS疾病的致病机制研究提供了新的思路。
关键词： hoxa1a基因;  ABDS;  颅骨发育;  CRISPR/Cas9    
Abstract:

hox genes encode a family of highly conserved transcription factors. Human HOXA1 mutation causes ABDS (athabascan brainstem dysgenesis syndrome), which leads to craniofacial bone deformity induced facial defect and paralysis. In this paper, zebrafish was used to study the functional mechanism of the homologous gene hoxa1a. Firstly, hoxa1a gene was edited by using CRISPR/Cas9 technology, which resulted in gene mutation. The T7E1 assay showed F0 digestion efficiency was 70% on average. Then F1 was screened and it was found that hoxa1a heterozygote generated 8 bp insertion and 7 bp deletion. Furthermore, the heterozygotes were crossed and hoxa1a homozygous F2 mutant was obtained, which was confirmed by sequencing. At 5 dpf, homozygous mutants of hoxa1a showed craniofacial dysplasia. The results of alcian blue cartilage staining and alizarin red hard bone staining demonstrated that the hoxa1a mutant had abnormal skull development, fracture of ethmoid plate, and defect of gill arch development. In this study, ABDS disease model in zebrafish was successfully constructed and the results indicate that hoxa1a mutation might cause abnormal craniofacial skeletal development, which lays a foundation for its mechanism study and provides a new idea for the pathogenesis of human ABDS disease.
Key words: hoxa1a gene    ABDS    Craniofacial skeletal development    CRISPR/Cas9
收稿日期: 2021-05-29 出版日期: 2021-09-30
ZTFLH:  Q812  
基金资助: * 国家自然科学基金(32170423);国家自然科学基金(31501166);上海市科委扬帆计划(15YF1405000)
通讯作者: 祖尧     E-mail: yzu@shou.edu.cn
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引用本文:

武秀知,王宏杰,祖尧. 斑马鱼hoxa1a基因调控颅面骨骼发育的功能研究*[J]. 中国生物工程杂志, 2021, 41(9): 20-26.

WU Xiu-zhi,WANG Hong-jie,ZU Yao. Functional Study of hoxa1a Regulating Craniofacial Bone Development in Zebrafish. China Biotechnology, 2021, 41(9): 20-26.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.2105054    或    https://manu60.magtech.com.cn/biotech/CN/Y2021/V41/I9/20

图1  利用CRISPR/Cas9系统编辑斑马鱼hoxa1a基因
图2  成功得到hoxa1a的纯合突变体斑马鱼,并发现面部骨骼发育畸形
图3  hoxa1a突变氨基酸序列及其蛋白质结构
图4  斑马鱼成鱼硬骨染色表明hoxa1a纯合突变体面部颅骨发育缺陷、筛骨板断裂
图5  hoxa1a缺失导致斑马鱼幼鱼面部软骨发育缺陷、筛骨板断裂
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