基因编辑酪氨酸酶(TYR)基因不同功能区对鱼类体色的影响

酪氨酸酶(Tyr)基因在动体的黑色素合成过程中起关键作用，其突变会导致人类和其他物种出现白化现象。本研究以AB系斑马鱼(Danio rerio)为研究对象，应用CRISPR/Cas9基因编辑技术，对纯合亲鱼受精卵进行显微注射，通过编辑损坏Tyr基因的CDS区第二外显子和3′-UTR非poly-A加尾信号区，使基因发生突变，检测基因不同功能区经编辑后对鱼类体色的影响。结果显示：Tyr基因在野生型斑马鱼的胚胎各个发育时期均能正常表达，眼睛出现黑色素时的表达量最高；编辑损坏Tyr基因的CDS区后，突变型斑马鱼的胚胎和仔鱼均未出现黑色素细胞，表现为体表白化，而成鱼会再度出现黑色素细胞，表现为体表黑色条纹断裂。编辑损坏Tyr基因的3′-UTR非poly-A加尾信号区后，突变型胚胎、仔鱼和成鱼均未出现黑色素减褪，黑色素合成未受影响。本研究表明，编辑损坏基因的CDS区会对生物表型产生显著影响，而编辑损坏3′-UTR非poly-A加尾信号区对生物表型的影响有限。

Fish coloration change from disrupting different functional regions in tyrosinase (TYR) gene by gene editing technology

In order to detect the effect of editing different functional regions of genes on fish coloration, fertilized eggs of AB strain of homozygous zebrafish (Danio rerio) were microinjected by CRISPR/Cas9 technology, Tyr gene were disrupted in the exon 2 and non-poly (A) addition signal of 3''-UTR, and mutants with disruption of different functional regions were obtained. The results showed that:the relative expression level of Tyr gene was detected in the examined stages of fertilized egg to 15 dph larvae in wild-type zebrafish, and the relative expression level increased to the highest level at melanin formation of eyes; the melanocytes were not observed in mutant embryo and larvae with disrupting the CDSregion of Tyr gene, displaying complete albino, then, the melanocytes were observed in adult fishes with broken melanocyte stripes. The mutants with disrupting non-poly (A) addition signal of 3''-UTR region of Tyr gene showed the same melanocyte stripes with wild-type zebrafish, indicating normal melanin synthesis in them. It was indicated that significantly phenotypic change would occur by disrupting CDS region of gene, while no obvious phenotypic change was detected by disrupting non-poly (A) addition signal of 3''-UTR region of gene.