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  

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

作　　者：冯田田 陶易凡 马昕羽[2] 路思琪 张幸[1] 刘文婷 潘奕凡 强俊 FENG Tiantian;TAO Yifan;MA Xinyu;LU Siqi;ZHANG Xing;LIU Wenting;PAN Yifan;QIANG Jun(School of Food Science and Pharmaceutical Engineering,Nanjing Normal University,Nanjing 210023,China;Wuxi Fisheries College,Nanjing Agricultural University,Wuxi 214081,China;Key Laboratory of Freshwater Fisheries and Germplasm Resources Utilization,Ministry of Agriculture,Freshwater Fisheries Research Center,Chinese Academy of Fishery Sciences,Wuxi 214081,China)

机构地区：[1]南京师范大学食品与制药工程学院,江苏南京210023 [2]南京农业大学无锡渔业学院,江苏无锡214081 [3]中国水产科学研究院淡水渔业研究中心,农业农村部淡水渔业与种质资源利用重点实验室,江苏无锡214081

出　　处：《中国水产科学》2023年第1期11-24,共14页Journal of Fishery Sciences of China

基　　金：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).

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(Oreochromisniloticus)的完整scdcDNA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scdcDNA序列全长1333bp,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(Daniorerio)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(highfatdietary,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scdmRNA表达量显著降低(P<0.05),而SCD^(-/-)组斑马鱼肝脏中lpl、fas、hslmRNA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。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 hi

关 键 词：吉富罗非鱼 硬脂酰辅酶A去饱和酶 基因克隆 斑马鱼 CRISPR/Cas9 脂质代谢 

分 类 号：S961[农业科学—水产养殖]