不同脂肪源饲料对斑马鱼elovl5、elovl2和fads2在高度不饱和脂肪酸合成中的作用影响

Effects of dietary different lipid sources on elovl5,elovl2 and fads2 in highly unsaturated fatty acids biosynthesis in zebrafish

为探究延长酶5(elovl5)、延长酶2(elovl2)和去饱和酶2(fads2)基因在淡水鱼高度不饱和脂肪酸(HUFA)合成中的相互作用,利用CRISPR/Cas9技术,成功构建出斑马鱼elovl5^(-/-)(以下简称E5^(-/-)组)、elovl2^(-/-)×elovl5^(-/-)(以下简称E2^(-/-)×E5^(-/-)组)、elovl5^(-/-)×fads2^(-/-)(以下简称E5^(-/-)×F2^(-/-)组)、elovl2^(-/-)×elovl5^(-/-)×fads2^(-/-)(以下简称E2^(-/-)×E5^(-/-)×F2^(-/-)组)4种突变体模型,并对4种突变体进行大豆油饲料(SO)和亚麻芥油饲料(CO)的投喂试验,研究斑马鱼elovl5、elovl2和fads2在HUFA合成过程中对饲料不同脂肪源的响应。结果显示,在E5^(-/-)组和E2^(-/-)×E5^(-/-)组中,C18PUFA未出现明显累积,而E5^(-/-)×F2^(-/-)组和E2^(-/-)×E5^(-/-)×F2^(-/-)组中的C18:3n-3(ALA)和C18:2n-6(LNA)呈现显著的累积。elovl2、elovl5和fads2缺失后,肝脏elovl4a和elovl4b表达明显上升。不同脂肪源饲料投喂试验结果显示,SO饲料投喂后的E5^(-/-)组DHA含量与野生型(WT)无显著差异,E5^(-/-)×F2^(-/-)组较WT组DHA含量显著降低。然而,CO饲料投喂后的E5^(-/-)组DHA含量较WT组呈现显著升高,E5^(-/-)×F2^(-/-)组与WT无显著差异。fads2缺失斑马鱼去饱和作用受到显著影响,表明,fads2是HUFA合成过程中的必需酶。而elovl2和elovl5缺失的表型可能会被其他HUFA合成途径所弥补。以上结果表明,elovl2和elovl5在HUFA合成中存在基因间相互作用,fads2作为去饱和酶在HUFA合成中必不可少。

In order to further explore the interaction of elongase 5(elovl5),elongase 2(elovl2)and desaturase 2(fads 2)in the synthesis of highly unsaturated fatty acids(HUFA)in freshwater fish,we successfully constructed four zebrafish mutations including elovl5^(-/-)(E5^(-/-)),elovl2^(-/-)×elovl5^(-/-)(E2^(-/-)×E5^(-/-)),elovl5^(-/-)×fads2^(-/-)(E5^(-/-)×F2^(-/-))and elovl2^(-/-)×elovl5^(-/-)×fads2^(-/-)(E2^(-/-)×E5^(-/-)×F2^(-/-))using CRISPR/Cas9 technology,and soybean oil(SO)and canola oil(CO)feeding experiments were carried out on four mutants.The responses of zebrafish elovl5,elovl2 and fads2 to different fat sources in the feed during the HUFA synthesis process were studied.The results showed that in the E5^(-/-)and E2^(-/-)×E5^(-/-)group,the extension of C18PUFA to C20PUFA still existed,while C18:3n-3(ALA)and C18:2n-6(LNA)showed significant accumulation in the E5^(-/-)×F2^(-/-)and E2^(-/-)×E5^(-/-)×F2^(-/-)group.The expression of elovl4a and elovl4b in the liver were induced after mutation of elovl2,elovl5 and fads2.The results of fatty acid analysis showed that the DHA content of E5^(-/-)group was not significantly different from that of WT,and the DHA content of E5^(-/-)×F2^(-/-)group was significantly lower than that of WT after fed with SO feed.However,the DHA content of E5^(-/-)group was significantly higher than that of WT,and there was no significant difference between E5^(-/-)×F2^(-/-)and WT after CO feed was fed.Zebrafish desaturation was significantly affected without fads2,indicating that fads2 is an essential enzyme in the process of HUFA synthesis.The missing phenotype of elovl2 and elovl5 may be compensated by other HUFA synthesis pathways.This experiment further verified that elovl2 and elovl5 had gene interactions in HUFA synthesis,and fads2 was essential in HUFA synthesis as a desaturase.This study provides a theoretical basis for improving the HUFA synthesis ability of freshwater fish.