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牙鲆(Paralichthys?olivaceus)tyrp1a和tyrp1b的鉴定及tyrp1a与mmu-miR-143-5p_R 2的调控关系研究
王若青,王娜,王仁凯,陈松林
作者单位E-mail
王若青 中国水产科学研究院黄海水产研究所 wangruoqing515@163.com 
王娜 中国水产科学研究院黄海水产研究所 wangna@ysfri.ac.cn 
王仁凯 中国水产科学研究院黄海水产研究所  
陈松林 中国水产科学研究院黄海水产研究所  
摘要:
牙鲆是我国北方重要的海水养殖鱼类,苗种生产中普遍存在体色白化现象,严重影响了苗种品质和市场价值。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microRNA (miRNA)深度测序数据的基础上,对tyrosinase related protein 1(Tyrp1)和mmu-miR-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的两个转录本,进化树分析表明这两个转录本分别是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的表达量明显低于白化牙鲆皮肤中的表达量。本研究发现牙鲆Tyrp1存在两个转录本,分别是Tyrp1a和Tyrp1b。双荧光素酶实验和定量PCR分析初步证实了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了重要的基础资料。
关键词:  牙鲆,白化,mmu-miR-143-5p_R 2,tyrp1a,tyrp1b
DOI:10.11758/yykxjz.20170508001
分类号:S917.4
基金项目:国家自然科学基金项目(31472273)、山东省重点研发计划(重大关键技术)项目(2016ZDJS06A07)和山东省泰山学者攀登计划项目共同资助 [This work was supported by the National Natural Science Foundation of China (31472273), Key R&D Program of shandong province (2016ZDJS06A07) and the Taishan Scholar Climbing Program of Shandong Province)].
The identification of tyrp1a and tyrp1b in Japanese Flounder (Paralichthys?olivaceus) and the regulation study of tyrp1a and mmu-miR-143-5p_R 2[ ① Corresponding author: Wang Na, E-mail: wangna@ysfri.ac.cn; Chen Songlin, E-mail: chensl@ysfri.ac.cn]
Wang Ruoqing,Wang Na,Wang Renkai,Chen Songlin
Abstract:
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. m Firstly, Tyrp1a and Tyrp1a were screened and identified by RACE and phylogenetic analysis. Subsequently, RNAhybrid 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 RT-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 were 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.
Key words:  Japanese flounder (Paralichthys?olivaceus), albinism, mmu-miR-143-5p_R 2, tyrp1a, tyrp1b