CRISPR/Cas9编辑Mlpha基因对斑马鱼黑色条纹形成的影响  

Effect on Black Stripes Formation in Zebrafish(Danio rerio)by CRISPR/Cas9 Editing on Mlpha Gene

作　　者：胡续雯 陈红林 余陆伟 许细丹 陈晓雯[1] 王军[1] 王成辉[1] HU Xu-Wen;CHEN Hong-Lin;YU Lu-Wei;XU Xi-Dan;CHEN Xiao-Wen;WANG Jun;WANG Cheng-Hui(Key Laboratory of Freshwater Aquatic Genetic Resources certificated by the Ministry of Agriculture and Rural Affairs/National Demonstration Center for Experimental Fisheries Science Education/Shanghai Engineering Research Center of Aquaculture,Shanghai Ocean University,Shanghai 201306,China)

机构地区：[1]上海海洋大学农业农村部淡水水产种质资源重点实验室/水产科学国家级实验教学示范中心/上海水产养殖工程技术研究中心,上海201306

出　　处：《农业生物技术学报》2021年第8期1453-1462,共10页Journal of Agricultural Biotechnology

基　　金：国家自然科学基金(31372521);广西科技重大专项(桂科AA17204095-7)。

摘　　要：黑素亲和素(melanophilin,Mlph)基因在脊椎动物的成熟黑素小体转运过程中具有关键作用,其突变会导致动物的毛色或羽色变淡等现象,但水产动物中的相关研究却极为薄弱。本研究以斑马鱼(Danio rerio)为研究对象,分析了Mlpha基因在斑马鱼不同发育阶段的表达情况,进而应用CRISPR/Cas9基因编辑技术对该基因进行了敲除,观察了F 2纯合突变体的表型变化,探讨了Mlpha基因对斑马鱼体色的影响。结果发现:Mlpha基因在斑马鱼的不同发育时期均能表达,且在出膜后5 d(即腹部出现较明显黑色素带时期)的表达量最高。编辑Mlpha基因后,嵌合型突变体的胚胎和幼鱼黑色素扩散缓慢,成鱼体表黑色条带断裂,部分黑色素细胞急剧缩小。F_(2)纯合突变体由于移码突变导致终止子出现,从而使Mlpha基因的蛋白翻译被截断,其成鱼体表的黑色素细胞也急剧缩小。进一步通过原位杂交和定量表达分析,发现与Mlpha基因相互作用的转运基因,即小GTP结合蛋白(member RAS oncogene family,Rab27a)和肌球蛋白Va(myosin Va,MyoVa)的表达量在纯合突变体中也显著降低(P<0.05)。本研究表明,Mlpha基因突变影响了黑色素的运输过程,从而导致斑马鱼黑色条纹的形成受阻。研究结果为了解鱼类黑斑体色形成的遗传基础与机制提供了有益的参考依据。Melanophilin(Mlph)gene plays fundamental roles in regulating the transportation of mature melanosomes in vertebrates,and its mutations can lead to hair or feather color fading in animals.However,the molecular function of Mlpha gene in aquatic animals need to be explored.In this study,spatial and temporal expression of Mlpha gene were investigated during different developmental stages in zebrafish(Danio rerio),and Mlpha gene was also knocked-out by using the CRISPR/Cas9 technology to exam body color changes in the F_(2) homozygous mutants in zebrafish.The results of in situ hybridization and real-time quantitative PCR(qRT-PCR)expression indicated that Mlpha gene expressed at all the collected developmental stages,including 6,12,24,30,36,48,60 hpf(hours post fetilization)and 1,2,3,4,5,10,15 dph(days post hatching)and the expression level increased with the increase of dendritic melanocytes in zebrafish.Meanwhile,the highest expression level was identified on the abdomen of zebrafish at 5 dph when obvious melanin band appeared.Similarly,the in situ hybridization results also showed the detectable signals and the strongest signal at 5 dph.After Mlpha gene was knocked-out by using CRISPR/Cas9 technology,the larval individuals of F_(1) mosaic mutant zebrafish displayed slow expansion of melanin in juveniles and fragmented black stripe in adults.Under observation using the steroscope,the numbers of melanocytes and their coloration were not significantly reduced at the stripe fragments,but their morphology was reduced and dendritic structure was degenerated.Furthermore,the development speed of Mlpha knockout zebrafish was slower than that of wild-type zebrafish,and the diffusion speed of melanin was also significantly slower than that of wild-type zebrafish,indicated that melanosome dispersion was affected if Mlpha gene was knocked-out.In the F 2 homozygous mutant zebrafish,the Mlpha protein could not be completely translated owing to a terminator codon production in the second exon of this gene,and the adults appeared s

关 键 词：黑素亲和素(Mlph)基因 黑素小体 CRISPR/Cas9 原位杂交 基因表达 

分 类 号：S917.4[农业科学—水产科学]