斑马鱼<i>tbx2b</i>调控心脏房室间隔发育的功能研究<sup>*</sup>

doi:10.13523/j.cb.2103060

中国生物工程杂志

2021, Vol. 41

Issue (8): 1-7 DOI: 10.13523/j.cb.2103060

研究报告

斑马鱼tbx2b调控心脏房室间隔发育的功能研究^*

赵霞^1,^2,³,朱哲^1,^2,³,祖尧^1,^2,^3,^**(

)

1 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
2 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
3 上海海洋大学国家水生动物病原库上海 201306

tbx2b Affects Atrioventricular Canal Development in Zebrafish

ZHAO Xia^1,^2,³,ZHU Zhe^1,^2,³,ZU Yao^1,^2,^3,^**(

)

1 International Research Center for Marine Biosciences, Ministry of Science and Technology, College of Fisheries and Life Sciences, Shanghai Ocean University, Shanghai 201306, China
2 Key Laboratory of Exploration and Utilization of Aquatic Genetic Resources, Ministry of Education, College of Fisheries and Life Sciences, Shanghai Ocean University, Shanghai 201306, China
3 National Pathogen Collection Center for Aquatic Animals, Shanghai Ocean University, Shanghai 201306, China

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摘要：

tbx2是早期心脏发育的关键基因。为进一步探究其对房室间隔(AVC)发育的影响,研究利用CRISPR/Cas9介导的基因敲除技术,成功构建了斑马鱼tbx2b突变体。通过T7E1酶切对其F₀进行敲除效率检测,结果显示平均敲除效率约为57.5%。F₁进一步筛选获得tbx2b杂合突变体,测序结果显示突变类型为11 bp碱基缺失的移码突变。tbx2b杂合子内交获得纯合子,tbx2b纯合突变体在5 dpf死亡并出现早期心脏环化异常表型。斑马鱼整胚原位杂交实验显示在3 dpf tbx2b纯合突变体中, 心脏腔室分化特异性标志基因nppa、nppb表达上调并异位表达在AVC,而AVC发育关键基因has2的表达消失。高效构建tbx2b突变体并初探其对下游基因的影响,为后续深入研究tbx2b对心脏AVC发育的作用奠定了基础,同时加深了人们对早期心脏调控网络的认识。

关键词： tbx2b; 心脏发育; 房室间隔; CRISPR

Abstract:

tbx2 plays an important role in heart development. To further investigate the gene function in the atrioventricular canal (AVC) development, tbx2b^-/- mutation zebrafish was successfully established using the CRISPR/ Cas9 mediated gene knockout technique. The knockout efficiency of F₀ was detected by T7E1 assay, and the result showed that the average knockout efficiency was about 57.5%. Sanger sequencing confirmed that tbx2b F₁ mutant had a-11bp-base deletion and caused frameshift mutation. Homozygous mutations were lethal in 5 dpf and early embryos suffered from cardiac looping abnormalities. In situ hybridization in 3 dpf tbx2b^-/- showed ectopic expression of nppa and nppb, which are chamber-specific marker genes, and significantly decreased expression of has2, which is AVC marker gene. tbx2b ^-/- mutant was efficiently constructed and effects on downstream genes were discussed, which laid a foundation for further research on the effect of cardiac AVC development and understanding of early cardiac regulatory networks.

Key words: tbx2b Heart development Atrioventricular canal CRISPR

收稿日期: 2021-03-23 出版日期: 2021-08-31

ZTFLH:

Q812

基金资助: * 国家自然科学基金(31501166);上海市教委晨光计划资助项目(14CG49)

通讯作者: 祖尧 E-mail: yzu@shou.edu.cn

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引用本文:

赵霞,朱哲,祖尧. 斑马鱼tbx2b调控心脏房室间隔发育的功能研究^*[J]. 中国生物工程杂志, 2021, 41(8): 1-7.

ZHAO Xia,ZHU Zhe,ZU Yao. tbx2b Affects Atrioventricular Canal Development in Zebrafish. China Biotechnology, 2021, 41(8): 1-7.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.2103060 或 https://manu60.magtech.com.cn/biotech/CN/Y2021/V41/I8/1

表1 斑马鱼tbx2b检测引物序列

表2 原位杂交探针序列

图1 斑马鱼tbx2b基因的进化地位

图2 CRISPR/ Cas9介导的基因编辑技术敲除斑马鱼tbx2b基因

图3 Tbx2b突变氨基酸序列及其蛋白质结构

图4 tbx2b纯合突变体表型观察及tbx2b原位杂交检测

图5 nppa、nppb和has2原位杂交结果

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