吉富罗非鱼硬脂酰辅酶A去饱和酶基因克隆、表达及其在斑马鱼中的转植研究
作者:
作者简介:

冯田田(1996–),女,硕士研究生,研究方向为水产生物技术.E-mail:ftt17865815919@163.com

中图分类号:

S961

基金项目:

国家自然科学基金项目(31502143; 32002363); 中国水产科学研究院基本科研业务费专项(2020TD37; 2021XT08).


Cloning and expression analysis of the stearoyl-CoA desaturase gene in the genetic improvement of farmed tilapia (Oreochromis niloticus) and transference analysis in zebrafish
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    摘要:

    硬脂酰辅酶 A 去饱和酶(stearoyl-CoA desaturates, SCD)是单不饱和脂肪酸合成的关键限速酶, 它在调节肝脏脂肪生成、脂肪酸代谢和脂质氧化方面发挥着重要作用。在本研究中, 利用 RACE 技术克隆了吉富罗非鱼 (Oreochromis niloticus)的完整 scd cDNA 序列, qRT-PCR 分析了组织表达特点。为了验证 scd 基因的功能, 利用 CRISPR/Cas9 技术构建 scd 基因敲除斑马鱼模型, 研究了 F3 突变体的表型和基因表达变化, 并结合高脂饲料实验验证了 scd 基因缺失后斑马鱼脂代谢的调控机制。结果显示, 吉富罗非鱼 scd cDNA 序列全长 1333 bp, 其中包括 173 bp、1008 bp、152 bp 的 5?非编码区、开放阅读框和 3′非编码区, 编码 335 个氨基酸。scd 基因在雄性和雌性罗非鱼的各组织中都有表达, 在肝脏内表达量最高, 脾脏内表达量最低。利用 CRISPR/Cas9 构建了 scd 基因敲除的斑马鱼(Danio rerio)模型进行功能验证, 与野生型[SCD(+/+)]斑马鱼相比, 纯合型[SCD(–/–)]斑马鱼腹部明显膨大。 Western blot 和 qRT-PCR 结果显示, 与 SCD(+/+)组斑马鱼相比, scd 基因在 SCD(–/–)组斑马鱼中表达显著降低(P<0.05), 且 SCD 蛋白在 SCD(–/–)组斑马鱼表达丰度也显著降低(P<0.05)。在高脂饲料(high fat dietary, HFD, 脂肪含量 16%) 投喂下, 与 HFD+SCD2(+/+)组斑马鱼相比, HFD+SCD3(–/–)组斑马鱼肝组织细胞中红色脂滴明显减少。qRT-PCR 结果显示, scd 基因敲除后, 与 SCD(+/+)组斑马鱼相比, SCD(–/–)组斑马鱼中 scd mRNA 表达量显著降低(P<0.05), 而 SCD(–/–)组斑马鱼肝脏中 lpl、fas、hsl mRNA 表达量均显著升高(P<0.05)。与投喂对照组饲料的斑马鱼相比, 高脂饲料投喂能显著升高其肝脏 scd mRNA 表达量与抑制 fas mRNA 表达量(P<0.05)。研究结果表明 scd 基因敲除可以缓解高脂饲料投喂下斑马鱼肝脏的脂肪沉积, scd 基因在鱼类脂肪酸代谢和脂质合成中可能发挥了重要作用。

    Abstract:

    With the rapid development of integrated farming of tilapia, various metabolic diseases such as nutritional fatty liver disease are becoming increasingly serious. Severe fatty liver can cause liver failure and cause death in tilapia. It is particularly important to understand the mechanism of lipid metabolism in patients with fatty liver disease. Zebrafish, as a model animal for developmental biology research, is also a research hotspot for liver disease models. Stearoyl-CoA desaturase (SCD) is a key rate-limiting enzyme for MUFA, which plays an important role in regulating hepatic lipogenesis, fatty acid metabolism, and lipid oxidation. Therefore, it is important to study the regulatory mechanism of the scd gene in lipid metabolism in fish. In this study, the complete scd cDNA sequence of GIFT was cloned by RACE and qRT-PCR was used to analyze tissue expression characteristics. To verify the function of scd, an scd knockout zebrafish model was constructed using CRISPR/ Cas9 technology, and the phenotypic and gene expression changes of F3 mutants were studied, and the response mechanism of zebrafish lipid metabolism after scd deletion was verified in combination with high-fat diet experiments. The results showed that the cDNA full-length sequence of the scd gene of GIFT was 1,333 bp in length, including 173 bp at the 5′-UTR, 152 bp at the 3′-UTR, and a 1,008 bp open reading frame (ORF) encoding 335 amino acids. The scd gene was expressed in all tissues of male and female GIFT, with the highest expression in the liver and the lowest expression in the spleen. An scd knockout zebrafish model was constructed using CRISPR/Cas9 for functional validation, and the SCD(–/–) zebrafish had a significantly enlarged abdomen compared to the SCD(+/+) zebrafish. Western blot and qRT-PCR results showed that scd gene expression was significantly lower (P<0.05) in zebrafish from the SCD(–/–) group compared with zebrafish from the SCD(+/+) group, and SCD protein expression abundance was also significantly lower (P<0.05) in zebrafish from the SCD(–/–) group. Under high fat dietary feeding, the red lipid droplets in the liver tissue cells of zebrafish in the HFD+SCD(–/–) group were significantly reduced compared to those in the HFD+SCD(+/+) group. The qRT-PCR results showed that scd mRNA expression was significantly lower in zebrafish in the SCD(–/–) group compared with zebrafish in the SCD(+/+) group after scd knockdown (P<0.05), while lpl, fas, and hsl mRNA expression were significantly higher in the liver of zebrafish in the SCD(–/–) group (P<0.05). Compared with the zebrafish fed the control diet, zebrafish fed with the high-fat diet had significantly increased expression of scd mRNA in liver and inhibited the expression of fas mRNA (P<0.05). The results also suggest that scd gene knockdown can alleviate liver fat deposition in zebrafish caused by high fat diets, and that the scd gene may play an important role in fatty acid metabolism and lipid synthesis in fish.

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冯田田,陶易凡,马昕羽,路思琪,张幸,刘文婷,潘奕凡,强俊.吉富罗非鱼硬脂酰辅酶A去饱和酶基因克隆、表达及其在斑马鱼中的转植研究[J].中国水产科学,2023,30(1):11-24
FENG Tiantian, TAO Yifan, MA Xinyu, LU Siqi, ZHANG Xing, LIU Wenting, PAN Yifan, QIANG Jun. Cloning and expression analysis of the stearoyl-CoA desaturase gene in the genetic improvement of farmed tilapia (Oreochromis niloticus) and transference analysis in zebrafish[J]. Journal of Fishery Sciences of China,2023,30(1):11-24

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