国家自然科学基金 (31702333)；中国水产科学研究院创新团队项目 (2020TD20)；国家现代农业产业技术体系专项 (CARS-47-G03)；山东省泰山学者攀登计划项目；中国水产科学研究院基本科研业务费专项 (2020XT0101)
National Natural Science Foundation of China (31702333); Innovation Team Project of China Academy of Fisheries Science (2020TD20) ; China Agriculture Research System （CARS-47-G03); Taishan Scholar Climbing Project of Shandong Province; Central Public-interest Scientific Institution Basal Research (2020XT0101)
为探究人工养殖半滑舌鳎无眼侧皮肤经常出现黑化现象的成因，实验根据本课题组前期全转录组数据分析结果预测得出lnc-XR_003049606.1与pmelb在基因层面上存在反式调控的关系。在半滑舌鳎无眼侧黑化皮肤中克隆获得pmelb的cDNA序列。生物信息学分析结果显示，pmelb的cDNA序列全长1 755 bp，编码584个氨基酸，其预测的蛋白分子量为63.94 ku，理论等电点为5.12，预测的二级蛋白结构含有1个Pleckstrin同源域、1个布鲁顿酪氨酸激酶富胱氨酸基序、1个Src同源3域、1个Src同源2域和1个酪氨酸激酶催化结构域。系统进化树分析显示， pmelb与其他鱼类对应的基因聚为一支。氨基酸同源性分析显示，半滑舌鳎的pmelb与大菱鲆和牙鲆的相似性最高 (分别为68.05%和66.28%)。分别通过分析半滑舌鳎lnc-XR_003049606.1和pmelb的表达分析发现，lnc-XR_003049606.1在1龄半滑舌鳎的皮肤和肝脏中表达，皮肤中表达量在60 d达到顶峰，随着时间延长而降低；而pmelb则主要在皮肤中表达，表达量在时间线上先升高再降低。本研究初步探究了lnc-XR_003049606.1和pmelb间的表达关系，为进一步筛选半滑舌鳎无眼侧黑化过程中起作用的关键基因提供了思路。
Fish coloration has long been a scientific research hotspot, owing not only to complex biological mechanisms associated with the prominent phenotypic characteristics, but also to high evolutionary significance for adaptation. Lately, an increasing number of researches have focused on lncRNA, due to their involvement in biological regulatory function. However, studies on lncRNAs in non-model organism such as Cynoglossus semilaevis are still lacking, particularly on skin melanostic mechanism. C. semilaevis is an important farmed marine fish in China and is easy to present the melanistic problem of blind side skin in the process of artificial cultivation. The blind side pigment abnormalities would spread with varying degrees of areas. It has not been known how pigment is correctly transported to the corresponding skin in C. semilaevis so far. In order to investigate the blind side melanized mechanism of lncRNA and mRNA in C. semilaevi, this experiment predicted that lnc-XR_003049606.1 targets pmelb through trans-acting according to the results of the transcriptome analysis. Bioinformatics analysis revealed that the full length of the cDNA of pmelb was 1 755 bp, encoding 584 amino acids, with a predicted protein molecular weight of 63.94 ku and a theoretical isoelectric point of 5.12. The predicted secondary protein structure contains a Pleckstrin homology domain, a Bruton's tyrosine kinase Cys-rich motif, a Src homology 3 domains, a Src homology 2 domains and a tyrosine kinase catalytic domain. Phylogenetic tree analysis showed that pmelb was clustered with corresponding genes in other fishes. The amino acid homology analysis showed that pmelb had the highest similarity with Scophthalmus maximus and Paralichthys olivaceus (68.05% and 66.28%, respectively). Analysis of the spatio-temporal expression patterns on lnc-XR_003049606.1 and pmelb revealed that lnc-XR_003049606.1 was expressed in the skin and liver of 1-year-old tongue sole, with expression peaking in the skin at 60 days and then decreasing over time, whereas pmelb was mainly expressed in the skin, with expression increasing and then decreasing over the timeline. This study provided an idea for further screening the key genes that play a role in the process of blind side hypermelanotic skin in C. semilaevi.