利用CRISPR/Cas9技术构建表达鲤长链脂肪酸延长酶5基因(Elovl5)的转基因斑马鱼  

Construction of Transgenic Zebrafish(Danio rerio)Expressing Common Carp(Cyprinus carpio)Elovl5 Using CRISPR/Cas9 System

作　　者：王雅欣 杨晨茹 赵宇杰 孙晓晴[2] 李尚琪 张研[2] 刘英杰[2] 李炯棠[2] WANG Ya-Xin;YANG Chen-Ru;ZHAO Yu-Jie;SUN Xiao-Qing;LI Shang-Qi;ZHANG Yan;LIU Ying-Jie;LI Jiong-Tang(National Experimental Teaching and Demonstration Center of Fisheries Science,Shanghai Ocean University,Shanghai 201306,China;Biotechnology Research Center,Chinese Academy of Fishery Sciences/Key Laboratory of Aquatic Genomes,Ministry of Agriculture and Rural Affairs/Beijing Key Laboratory of Aquatic Biotechnology,Beijing 100141,China)

机构地区：[1]上海海洋大学水产科学国家级实验教学示范中心,上海201306 [2]中国水产科学研究院生物技术研究中心/农业农村部水生动物基因组学重点实验室/北京市渔业生物技术重点实验室,北京100141

出　　处：《农业生物技术学报》2020年第7期1165-1176,共12页Journal of Agricultural Biotechnology

基　　金：国家自然科学基金(31672644);国家重点研发计划(2018YFD0900102);中国水产科学研究院级基本业务费(2018HY-ZD0207;2018B004)。

摘　　要：CRISPR/Cas9技术广泛应用于基因敲除,利用敲入包含荧光蛋白和目标基因的片段,有助于早期筛选实验动物。长链脂肪酸延长酶5(elongation of very long chain fatty acids 5,Elovl5)是鱼类调控高不饱和脂肪酸合成的关键酶之一。为在斑马鱼(Danio rerio)研究鲤(Cyprinus carpio)Elovl5活性,本研究构建包含Tol2序列、斑马鱼短链肌球蛋白2基因(myosin light chain 2,Mylz2)启动子、红色荧光蛋白基因(red fluorescence gene,DsRed2)、斑马鱼Elovl5启动子、鲤Elovl5基因、2A剪切肽和绿色荧光蛋白(enhanced green fluorescent protein,EGFP)的载体,扩增获得一段长7.5 kb的片段。利用CRISPR/Cas9方法,将此片段敲入斑马鱼基因组。敲入成功的斑马鱼在受精72 h后开始于肌肉显红色荧光。对利用红色荧光筛选敲入成功的斑马鱼,进一步用PCR扩增DsRed2予以确证。在存活斑马鱼中,基因敲入阳性率为28.7%。相比对照组,仅在实验组观察到鲤Elovl5表达。实验组的18碳不饱和脂肪酸含量下降18.6%,20碳和22碳不饱和脂肪酸含量提高31.8%,其中20:4n-3不饱和脂肪酸含量是对照组的2.4倍(P<0.05)。本研究采用CRISPR/Cas9技术,构建转鲤Elovl5斑马鱼,提高了斑马鱼高不饱和脂肪酸含量,为进一步研究鲤不饱和脂肪酸合成的遗传机制提供基础。CRISPR/cas9 technology is widely used in gene knockout.It is helpful for early screening of experimental animals to knock in fragments containing fluorescent protein and target gene.Elongation of very long chain fatty acids 5(Elovl5)is one of key enzymes to regulate the biosynthesis of fish highly-unsaturated fatty acids(HUFAs).Constructing a transgenic animal model expressing Elovl5 will provide a new strategy to study the in vivo function of this enzyme.Common carp(Cyprinus carpio)is one of the most widely cultured freshwater fishes in our country.Although common carp Elovl5 was identified and its tissue expressional pattern and correlation to growth were studied,its elongase activity has not been investigated.To study the in vivo function of common carp Elovl5 in zebrafish(Danio rerio),a vector including Tol2 sequence,zebrafish myosin light chain 2(Mylz2)promoter,red fluorescence gene(DsRed2),zebrafish Elovl5 promoter,common carp Elovl5 gene,2A cleavage peptide,and green fluorescence gene,was constructed.A fragment consisting of these promoters and genes with a length of 7.5 kb was amplified from this vector and then knocked into zebrafish genome using the CRISPR/Cas9 method.From 72 h after fertilization,the red fluorescence was observed.For the transgenic zebrafish screened using the red fluorescence,PCR amplification of DsRed2 confirmed the insertion of this fragment into the genome.The result indicated that using fluorescence to screen transgenic animals was reliable,simple and practicable.Among all the surviving zebrafish,the knock-in rate was 28.7%.The study also knocked this fragment into the genome by using Tol2 system with a rate of 32.8%,which suggested that the knock-in efficiency of CRISPR/Cas9 method was lower than the Tol2 system.Compared to the control group,common carp Elovl5 was expressed only in the CRISPR/Cas9 experimental group and the Tol2 experimental group.Furthermore,its expression level was comparable between these two groups,suggesting that the inserted gene expression efficiency of th

关 键 词：长链脂肪酸延长酶5 (Elovl5) CRISPR/Cas9 基因敲入 鲤 高不饱和脂肪酸 

分 类 号：S917.4[农业科学—水产科学] S961.6