牙鲆变态中甲状腺激素调控的靶基因  

Target genes regulated by thyroid hormone during metamorphosis of Paralichthys olivaceus

作　　者：陶家康 付元帅[1,2,3] 施志仪[1,2,3] TAO Jiakang;FU Yuanshuai;SHI Zhiyi(Key Laboratory of Freshwater Aquatic Genetic Resources,Ministry of Agriculture and Rural Affair,Shanghai Ocean University,Shanghai 201306,China;Key Laboratory of Exploration and Utilization of Aquatic Genetic Resources,Ministry of Education,Shanghai Ocean University,Shanghai 201306,China;Shanghai Collaborative Innovation for Aquatic Animal Genetics and Breeding,Shanghai Ocean University,Shanghai 201306,China)

机构地区：[1]上海海洋大学,农业农村部淡水水产种质资源重点实验室,上海201306 [2]上海海洋大学,教育部水产遗传资源开发利用重点实验室,上海201306 [3]上海海洋大学,上海水生动物遗传育种协同创新中心,上海201306

出　　处：《中国水产科学》2020年第9期1010-1020,共11页Journal of Fishery Sciences of China

基　　金：国家自然科学基金项目(41676138,41506159)。

摘　　要：本研究旨在采用新的方法找出甲状腺激素受体(TRs)介导的甲状腺激素(TH)调控的靶基因。以牙鲆(Paralichthys olivaceus)为实验样本,表达并纯化了牙鲆p3×Flag-TRαA和p3×Flag-TRβ融合蛋白,采用一种目的基因循环扩增与筛选的方法,寻找TH直接调控的靶基因,最终筛选得到TRαA的候选靶基因11个,TRβ的候选靶基因12个,其中TRαA和TRβ存在4个相同的候选靶基因:frizzled-3-like、TBC1结构域激酶(tbck)、氨酰tRNA合成酶复合多功能相互作用蛋白1(aimp1)和MYC相关因子X(max)。利用荧光定量PCR技术检验了这4个基因在牙鲆不同组织、不同变态发育期以及外源性甲状腺激素和硫脲处理后各个变态发育时期的表达情况,发现在TH含量升高时,靶基因表达量降低;TH含量降低时,靶基因的表达量上升,从而验证靶基因受到TH的负向调控,证实TH在促进牙鲆变态中也存在抑制靶基因转录的情况。本研究旨为研究TH调控牙鲆变态的发育网络提供基础资料。There are large differences in morphology and living habits of Japanese flounder(Paralichthys olivaceus)in the early stages of metamorphic development.These differences make P.olivaceus an ideal model of metamorphosis in fish.The thyroid hormone(TH)plays a decisive role in the development of metamorphosis.The physiological role of TH is to regulate the transcription of target genes via the thyroid hormone receptors(TRs)and thyroid hormone response elements(TRE)—AGGT(C/A)A.In this study,P.olivaceus was used to investigate a new method for finding the target genes regulated by TH.We expressed and purified the p3×Flag-TRαA and p3×Flag-TRβfusion proteins to identify the target genes downstream of TH by using a cyclic amplification and selection of target(CAST)method.We found 11 potential TRαA target genes and 12 potential TRβtarget genes.TRαA and TRβshare 4 identical potential target genes,which are frizzled-3-like,TBC1 domain-containing kinase(tbck),aminoacyl tRNA synthetase complex interacting multifunctional protein 1(aimp1),and MYC associated factor X(max).The expression of these genes was tested in different adult tissues and different metamorphosis development stages of P.olivaceus in the NC(normal control),TH(thyroid hormone),and TU(thiourea)groups by real-time quantitative PCR.The expression of these genes was highest in the adult brain tissue(P<0.05).The expression of these genes during early metamorphic development was highest at 17 dph(days post hatching)and lowest at 28 dph and 32 dph.The expression of these genes in the TH group was lower than in the NC group(P<0.05).The difference between the NC group and the TH group at 17 dph was the largest(P<0.05),while NC and TH groups had the smallest expression differences at 28 dph and 32 dph(P>0.05).Compared to the TH group,the expression in the TU group increased slightly in each period(P>0.05).These results indicate that frizzled-3-like,tbck,aimp1,and max are target genes directly regulated by TH and that the expression of these genes is inhibited b

关 键 词：牙鲆 甲状腺激素 甲状腺激素受体 DNA-蛋白质互作 靶基因 

分 类 号：S961[农业科学—水产养殖]