敲除hdac11基因对斑马鱼脂代谢的影响

doi:10.19586/j.2095-2341.2023.0042

生物技术进展 ›› 2023, Vol. 13 ›› Issue (4): 588-595.DOI: 10.19586/j.2095-2341.2023.0042

• 研究论文 • 上一篇

敲除hdac11基因对斑马鱼脂代谢的影响

熊茂兰¹(), 蔚思言², 罗军涛¹, 韩兵社¹, 张俊芳¹()

^1.上海海洋大学水产与生命学院，水产种质资源发掘与利用教育部重点实验室，水产科学国家级实验教学师范中心，上海 201306
^2.上海海洋大学海洋科学学院，上海 201306

收稿日期:2023-03-30 接受日期:2023-04-28 出版日期:2023-07-25 发布日期:2023-08-03
通讯作者: 张俊芳
作者简介:熊茂兰 E-mail: 1023968325@qq.com；
基金资助:
上海市科技兴农重点攻关项目(沪农科创字〔2019〕第1-2号)

The Effects of hdac11 Knockout of Zebrafish on Lipid Metabolism

Maolan XIONG¹(), Siyan WEI², Juntao LUO¹, Bingshe HAN¹, Junfang ZHANG¹()

^1.National Demonstration Center for Experiment Fisheries Science Education，Key Laboratory of Exploration and Utilization of Aquatic Genetic Resources，Ministry of Education，College of Fisheries and Life Science，Shanghai Ocean University，Shanghai 201306，China
^2.College of Marine Science，Shanghai Ocean University，Shanghai 201306，China

Received:2023-03-30 Accepted:2023-04-28 Online:2023-07-25 Published:2023-08-03
Contact: Junfang ZHANG

摘要/Abstract

摘要：

为揭示斑马鱼组蛋白去乙酰化酶11（histone deacetylase 11，HDAC11）在斑马鱼脂代谢中的作用，利用CRISPR/Cas9技术成功构建斑马鱼hdac11^-/- 模型，将受精5 d后(5 days post fertilization，5 dpf)的WT与hdac11^-/- 斑马鱼分别以正常饮食和高脂饮食2种方式饲喂至90 dpf，检测斑马鱼体重和体长，观察肝脏组织学切片并检测肝脏甘油三酯含量和脂代谢相关基因表达水平。结果显示，与WT相比，hdac11^-/- 斑马鱼体重、体长无显著性差异；幼鱼时期正常饮食下hdac11^-/- 斑马鱼全鱼甘油三酯含量显著降低(P<0.001)；成鱼时期正常饮食和高脂饮食下hdac11^-/- 斑马鱼肝脏脂滴变小且肝脏甘油三酯含量均显著降低(P<0.05)；肝脏中pnpla2、lipeb基因表达水平均显著升高，gpam基因表达水平均显著下降。结果表明，hdac11通过改变甘油三酯合成与分解的基因表达水平进而影响脂代谢调控，这为鱼类脂代谢过程中的表观调控机制提供了新的研究方向。

关键词: hdac11, 斑马鱼, CRISPR/Cas9, 脂质代谢

Abstract:

To explore the function of histone deacetylase 11 （HDAC11） of zebrafish in lipid metabolism， we generated a hdac11^-/- zebrafish mutant line with CRISPR/Cas9 technology. We fed WT zebrafish and hdac11^-/- zebrafish with standard diet and high fat diet from 5 days post fertilization （dpf） to 90 dpf， measured their body weights and body lengths， observed histological sections and detected hepatic triglyceride content and lipid metabolism related gene expression of zebrafish. The results showed that there was no significant difference in body weight and body length betweeen hdac11^-/- mutants and WT zebrafish. Triglyceride content was significantly decreased in hdac11^-/- zebrafish compared to WT （P<0.001） at larval stage. The hepatic triglyceride content of adult zebrafish was significantly reduced under standard diet and high fat diet， and the liver lipid droplets were smaller. The expression levels of pnpla2 and lipeb were significantly increased， while the expression level of gpam was significantly decreased in liver. The results indicated that hdac11 participates in the lipid metabolic process in zebrafish and affects the regulation of lipid metabolism by changing the expression levels of genes involved in triglyceride synthesis and decomposition， which provides a new research direction for the epigenetic regulation mechanism to lipid metabolism in fish.

Key words: hdac11, zebrafish, CRISPR/Cas9, lipid metabolism

中图分类号:

S917.4

熊茂兰, 蔚思言, 罗军涛, 韩兵社, 张俊芳. 敲除hdac11基因对斑马鱼脂代谢的影响[J]. 生物技术进展, 2023, 13(4): 588-595.

Maolan XIONG, Siyan WEI, Juntao LUO, Bingshe HAN, Junfang ZHANG. The Effects of hdac11 Knockout of Zebrafish on Lipid Metabolism[J]. Current Biotechnology, 2023, 13(4): 588-595.

图/表 7

表1 实验所用引物序列

Table 1 Primer sequences used in this study

引物名称	序列（5′→3′）	碱基数/bp
hdac11-F	5′-CCTTGGCTCACCGTGTATTT-3′	20
hdac11-R	5′-AATTCCCACCCAGTATCATTGG-3′	22
RT-gpam-F	5′-CAGAGGCTCTTTCTTGGCGA-3′	20
RT-gpam-R	5′-CAGCTGATGGGCTCACCTTT-3′	20
RT-pnpla2-F	5′-AACGCGCGTCATATTTACGG-3′	20
RT-pnpla2-R	5′-CGCCGGCTTCTCCTAAACAG-3′	20
RT-lipeb-F	5′-GAAGTCTGGGAGCGGTGTTC-3′	20
RT-lipeb-R	5′-CTGCCTTGGTGTCCATCACTAT-3′	22
β-actin-F	5′-CTTTGAGCAGGAGATGGGAACC-3′	22
β-actin-R	5′-GATTCCATACCCAGGAAGG-3′	19

图1 利用CRISPR/Cas9构建hdac11突变体A：hdac11基因中CRISPR/Cas9靶点的位置，红色表示靶点序列，蓝色表示PAM区；B：F2代凝胶电泳图，+/+ 表示野生型， +/- 表示杂合子，-/-表示纯合突变；C：预测HDAC11蛋白的截短型；D：Western blot检测HDAC11蛋白的表达量变化

Fig. 1 Generation of hdac11 mutant using CRISPR/Cas9 system

图2 hdac11-/- 斑马鱼表型特征A：不同时间点体重（n=5）；B：不同时间点体长；C：斑马鱼表型。ns表示在P>0.05水平上无统计学意义。

Fig. 2 Phenotypic characterization of zebrafish WT and hdac11-/-

图3 斑马鱼幼鱼甘油三酯检测A：幼鱼油红O染色结果(×20），黑色箭头表示脂滴，蓝色箭头表示肝脏部位；B：幼鱼整鱼甘油三酯含量检测结果（n=9）,***表示差异在P<0.01水平有统计学意义。

Fig. 3 Triglyceride detection in larval zebrafish

图4 斑马鱼成鱼甘油三酯检测A：肝脏组织学染色（×200），Nu表示细胞核，Ld表示脂滴；B：肝脏甘油三酯含量检测（n=9）,*表示差异在P<0.05水平有统计学意义。

Fig. 4 Triglyceride detection in adult zebrafish.

图5 高脂诱导下hdac11-/- 斑马鱼表型检测A：高脂诱导后体重变化；B：高脂诱导后体长变化；C：肝脏组织学染色(×400），Nu表示细胞核，Va表示空泡，Ld表示脂滴；D：肝脏甘油三酯含量检测（n=9）

Fig. 5 Phenotypic characterization in WT and hdac11-/- zebrafish under high fat diet

图6 不同饮食条件下肝脏甘油三酯相关基因表达检测A：SD时肝脏甘油三酯相关基因的表达；B：HFD时肝脏甘油三酯相关基因的表达

Fig. 6 Expression of genes related to triglyceride under different dietary conditions

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敲除hdac11基因对斑马鱼脂代谢的影响

The Effects of hdac11 Knockout of Zebrafish on Lipid Metabolism

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