Cloning and Preliminary Functional Study of the OCA2 Gene in Oujiang Color Common Carp (Cyprinus carpio var. color)
ZENG Chun-Xiao1,2,3, KE Jing1,2,3, ZHANG Peng-Hui1,2,3, CHEN Hui-Fan1,2,3, CHEN Xiao-Wen1,2,3, WANG Jun1,2,3, WANG Cheng-Hui1,2,3,*
1 Key Laboratory of Freshwater Aquatic Genetic Resources Certificated by the Ministry of Agriculture and Rural Affairs, Shanghai Ocean University, Shanghai 201306, China; 2 National Demonstration Center for Experimental Fisheries Science Education, Shanghai Ocean University, Shanghai 201306, China; 3 Shanghai Engineering Research Center of Aquaculture, Shanghai Ocean University, Shanghai 201306, China
Abstract：The OCA2 (oculocutaneous albinism Ⅱ) gene is an important gene in melanin biosynthesis, and involved in pigmentation in the eye, skin, and hair colors of animals. Mutations in this gene could result in oculocutaneous albinism type Ⅱ in human. The black patchs on the body surface are key phenotypic characteristics in Oujiang color common carp (Cyprinus carpio var. color), however, the genetic basis and underlying mechanisms of their formation remain unclear. In this study, The high-generation selected strains of Oujiang color common carp was used to perform the cloning, expression and promoter transcriptional activity studies of the OCA2 gene, and gene knockout experiment was carried out by the CRISPR/Cas9 technology. The results revealed that the OCA2 gene consisted of 23 exons and 22 introns, encoding 834 amino acids with over 97% homology with the other fish species in Cyprinid family, which indicated highly structural and functional conservation. The qPCR analysis of the OCA2 gene showed that its expression levels were significantly higher in the eye and skin tissues in the whole white with black patch than those in the whole white color types in Oujiang color common carp. The dual-luciferase reporter assays indicated a complex regulatory mechanism in the promoter region of the OCA2 gene, and a key regulatory element during the 600~1 200 bp fragment before the start codon. CRISPR/Cas9-mediated knockout of exon and promoter regions in the OCA2 gene resulted in the numbers reduction by 70% and the areas decreasing by 40% of melanocytes from surface skin and eye tissues in the mutant. This study revealed that the OCA2 gene plays an important role in pigmentation in Oujiang color common carp.
曾春晓, 柯景, 张鹏辉, 陈慧帆, 陈晓雯, 王军, 王成辉. 瓯江彩鲤OCA2基因的克隆与初步功能研究[J]. 农业生物技术学报, 2025, 33(12): 2668-2676.
ZENG Chun-Xiao, KE Jing, ZHANG Peng-Hui, CHEN Hui-Fan, CHEN Xiao-Wen, WANG Jun, WANG Cheng-Hui. Cloning and Preliminary Functional Study of the OCA2 Gene in Oujiang Color Common Carp (Cyprinus carpio var. color). 农业生物技术学报, 2025, 33(12): 2668-2676.
[1] 胡续雯. 2021. Mlpha基因对瓯江彩鲤和斑马鱼黑斑体色的影响[D]. 硕士学位论文, 上海海洋大学, 导师: 王成辉, pp. 20-27. (Hu X W.2021. Effect of melanophilin gene on black coloration in Oujiang color common carp (Cyprinus carpio var. color) and Zebrafish (Danio rerio) period[D]. Thesis for M.S., Shanghai Ocean University, Supervisor: Wang C H, pp. 20-27.) [2] 蒋月雯, 潘启华, 王志, 等. 2024. 基于密码子优化的SaCas9系统高效编辑青鳉(Oryzias latipes)基因组[J]. 浙江大学学报(英文版)(B辑: 生物医学和生物技术), 25: 1083-1096. (Jiang Y W, Pan Q H, Wang Z, et al.2024. Efficient genome editing in medaka (Oryzias latipes) using a codon-optimized SaCas9 system[J]. Journal of Zhejiang University-Science B (Biomedicine & Biotechnology), 25: 1083-1096.) [3] 司周旋, 陈红林, 许细丹, 等. 2020. 多巴色素异构酶基因对瓯江彩鲤黑斑体色的影响[J]. 中国水产科学, 27: 605-612. (Si Z X, Chen H L, Xu X D, et al.2020. Effect of Dct on pigmentation patterns in Oujiang color common carp[J]. Journal of Fishery Sciences of China, 27: 605-612.) [4] 王成辉, 项松平, 吕耀平, 等. 2008. 瓯江彩鲤红、白两种体色遗传关系的初步研究[J]. 上海海洋大学学报, 17: 402-405. (Wang C H, Xiang S P, Lv Y P, et al.2008. Preliminary study on inheritance of red and white color phenotypes in Oujiang color carp[J]. Journal of Shanghai Ocean University, 17: 402-405.) [5] 许细丹. 2020. 酪氨酸酶对瓯江彩鲤和斑马鱼黑斑体色影响的研究[D]. 硕士学位论文, 上海海洋大学, 导师: 王成辉, pp. 20-27. (Xu X D.2020. Study on the effect of Tyrosinase on black coloration in Oujiang color common carp and zebrafish[D]. Thesis for M.S., Shanghai Ocean University, Suppervisor: Wang C H, pp. 28-31.) [6] 许细丹, 陈红林, Kumar M B, 等. 2020. 基因编辑酪氨酸酶(TYR)基因不同功能区对鱼类体色的影响[J]. 上海海洋大学学报, 29: 811-819. (Xu X D, Chen H L, Kumar M B, et al.2020. Fish coloration change from disrupting different functional regions in tyrosinase (TYR) gene by gene editing technology[J]. Journal of Shanghai Ocean University, 29: 811-819.) [7] 喻明慧. 2022. 基于全基因组关联分析的黄河鲤体色异常分子机制及oca2等重要基因的功能初探[D]. 硕士学位论文, 上海海洋大学, 导师: 江炎亮, pp. 19-21. (Yu M H.2022. Genome-wide association study on the molecular mechanism underlying abnormal body color of Yellow River carp and the functions of oca2 and other important genes[D]. Thesis for M.S., Shanghai Ocean University, Supervisor: Jiang Y, pp. 19-21.) [8] Beirl A J, Linbo T H, Cobb M J, et al.2014. oca2 regulation of chromatophore differentiation and number is cell type specific in zebrafish[J]. Pigment Cell & Melanoma Research, 27: 178-189. [9] Bellono N W, Escobar I E, Lefkovith A J, et al.2014. An intracellular anion channel critical for pigmentation[J]. Elife, 3: e04543. [10] Brilliant M H.2001. The mouse p (pink‐eyed dilution) and human P genes, oculocutaneous albinism type 2 (OCA2), and melanosomal pH[J]. Pigment Cell & Melanoma Research, 14: 86-93. [11] Cheli Y, Ohanna M, Ballotti R, et al.2010. Fifteen‐year quest for microphthalmia‐associated transcription factor target genes[J]. Pigment Cell & Melanoma Research, 23: 27-40. [12] Chen H, Wang J, Du J, et al.2019. ASIP disruption via CRISPR/Cas9 system induces black patches dispersion in Oujiang color common carp[J]. Aquaculture, 498: 230-235. [13] Clark B, Elkin J, Marconi A, et al.2022. Oca2 targeting using CRISPR/Cas9 in the Malawi cichlid Astatotilapia calliptera[J]. Royal Society Open Science, 9: 220077. [14] Fontanesi L.2022. Invited review: Genetics and genomics of pigmentation variability in pigs: A review[J]. Livestock Science, 265: 105079. [15] Jing K, Chen H, Kanika N H, et al.2025. Qualitative and quantitative characteristics of black patch trait in Oujiang color common carp[J]. Aquaculture Reports, 40: 102619. [16] Klaassen H, Wang Y, Adamski K, et al.2018. CRISPR mutagenesis confirms the role of oca2 in melanin pigmentation in Astyanax mexicanus[J]. Developmental Biology, 441: 313-318. [17] Li S, Wang C.2001. Genetic diversity and selective breeding of red common carps in China[J]. The World Fish Center Quarterly, 24(3-4): 56-61. [18] Makova K, Norton H.2005. Worldwide polymorphism at the MC1R locus and normal pigmentation variation in humans[J]. Peptides, 26: 1901-1908. [19] Mesdaghi S, Murphy D L, Simpkin A J, et al.2023. Structural insights into pink-eyed dilution protein (Oca2)[J]. Bioscience Reports, 43: BSR20230060. [20] Oetting W S, King R A.1999. Molecular basis of albinism: Mutations and polymorphisms of pigmentation genes associated with albinism[J]. Human Mutation, 13: 99-115. [21] Rinchik E M, Bultman S J, Horsthemke B, et al.1993. A gene for the mouse pink-eyed dilution locus and for human type Ⅱ oculocutaneous albinism[J]. Nature, 361: 72-76. [22] Szczygieł J, Kamińska-Gibas T, Petit J, et al.2021. Re-evaluation of common carp (Cyprinus carpio L.) housekeeping genes for gene expression studies-considering duplicated genes[J]. Fish & Shellfish Immunology, 115: 58-69.
