牙鲆tyrp1a和tyrp1b的鉴定及tyrp1a与mmu-miR-143-5p_R+2的调控关系

The Identification of tyrp1a and tyrp1b in Japanese Flounder(Paralichthys olivaceus) and the Regulation Study of tyrp1a and mmu-mi R-143-5p_R+2

为了研究牙鲆白化发生过程中的分子调控机制,本研究在获得正常和白化牙鲆转录组及micro RNA(mi RNA)深度测序数据的基础上,对tyrosinase related protein 1(tyrp1)和mmu-mi R-143-5p_R+2(mmu-143)进行了表达模式、靶基因预测及验证分析。首先通过RACE方法克隆得到白化相关基因tyrp1的2个转录本,进化树分析表明这2个转录本分别是tyrp1a和tyrp1b,利用RNAhybrid软件预测到mmu-143可能与tyrp1a基因存在互作关系,通过双荧光素酶实验初步验证了这一靶向关系。进一步的荧光定量结果显示,tyrp1a基因在正常牙鲆皮肤中的表达量显著高于白化牙鲆皮肤的表达量,正常牙鲆皮肤中mmu-143的表达量显著低于白化牙鲆皮肤中的表达量。本研究发现,牙鲆tyrp1存在2个转录本,分别是tyrp1a和tyrp1b。双荧光素酶实验和定量PCR分析初步证实,tyrp1a是mmu-143的靶基因,mmu-143是通过调控tyrp1a基因的表达来影响牙鲆白化的。此研究结果为深入揭示牙鲆白化发生的分子机制提供重要的基础资料。

The albinism in Japanese flounder aquaculture has become a common phenomenon and influenced its large-scale farming and market value.Based on the albinism and normal Japanese flounder transcriptome and miRNA sequencing,tyrosinase related protein 1(tyrp1)and mmu-miR-143-5p_R+2(mmu-143)were chosen for expression pattern,target gene prediction and verification analysis.Firstly,tyrp1a and tyrp1b were screened and identified by RACE and phylogenetic analysis.Subsequently,RNA hybrid was used for the prediction of the targeting relationship between tyrp1a and miRNA mmu-143,which was further verified by dual luciferase experiment.Finally,the results of the quantitative real time-PCR(qRT-PCR)showed that the expression of tyrp1a gene in normal skin of Japanese flounder was significantly higher than the albinism skin,and the expression of mmu-143 in normal skin was significantly lower than the albinism skin.The present study identified two transcripts of tyrp1a and tyrp1b from Japanese flounder.Dual luciferase experiment and qRT-PCR analysis confirmed that tyrp1a was the target gene of mmu-143,and mmu-143 affected the albino of Japanese flounder by regulating tyrp1a.The results of this study will be helpful to fully understand the molecular mechanism of Japanese flounder albinism.