文蛤甲状腺激素受体MmTR互作蛋白筛选及其对Aroclor 1254胁迫的转录响应研究  

Screening of MmTR Interacting Proteins of Meretrix meretrix and Transcriptional Response to Aroclor 1254 Stress

作　　者：蔡月凤 谭皓文 吴钰泽 胡杰 姬南京 申欣 Cai Yuefeng;Tan Haowen;Wu Yuze;Hu Jie;Ji Nanjing;Shen Xin(School of Marine Science and Fisheries,Jiangsu Ocean University,Lianyungang 222005,China;Jiangsu Key Laboratory of Marine Bioresources and Environment/Jiangsu Key Laboratory of Marine Biotechnology,Lianyungang 222005,China;Co-Innovation Center of Jiangsu Marine Bio-industry Technology,Lianyungang 222005,China)

机构地区：[1]江苏海洋大学海洋科学与水产学院,连云港222005 [2]江苏省海洋生物资源与环境/海洋生物技术重点实验室,连云港222005 [3]江苏省海洋生物产业技术协同创新中心,连云港222005

出　　处：《生态毒理学报》2023年第3期296-311,共16页Asian Journal of Ecotoxicology

基　　金：江苏省农业科技自主创新资金资助项目[CX(21)3156];国家自然科学基金资助项目(41706135)。

摘　　要：环境中存在的甲状腺干扰物(thyroid disrupting chemicals,TDCs)可影响生物体内甲状腺激素(thyroid hormone,THs)的合成、分泌、转运、代谢和作用等过程,干扰THs稳态,引起THs水平失衡。甲状腺激素受体(thyroid hormone receptors,TRs)是一种存在于细胞核内的DNA结合转录因子,属于配体依赖型核激素受体超家族。TRs可与甲状腺激素响应元件(thyroid hormone response elements,TREs)结合,调控其转录表达。TDCs可作为生物体内TRs的配基,竞争性地与TRs结合,进而激活甲状腺信号通路,调控机体的整个甲状腺信号通路基因的转录表达。为探究TDCs对海洋无脊椎动物体内甲状腺激素是否具有干扰效应及其机制,本文通过构建文蛤消化盲囊组织酵母双杂交cDNA文库和文蛤甲状腺激素受体TR基因的诱饵载体pGBKT7-MmTR,在文库内进行MmTR的互作蛋白筛选和鉴定。结果表明,文蛤消化盲囊酵母双杂交cDNA文库中,cDNA片段大小在0.5~2 kb之间,库容约>2×10^(6) cfu。重组质粒pGBKT7-MmTR诱饵载体无毒性和自激活活性,可应用于酵母双杂交系统。经文库筛选共得到36个阳性克隆子,测序获得30个阳性克隆片段。随机选取BIRC2_3与MmTR基因进行点对点验证,结果为阳性,推测所有结果真实可靠。经比对分析,MmTR的29个潜在互作蛋白,功能涉及生长与发育过程、蛋白质代谢与免疫调控过程、应激响应过程和转录调控过程。不同浓度Aroclor 1254胁迫下幼体及成体文蛤体内MmTR基因潜在互作蛋白基因的转录响应分析发现,大多数潜在互作蛋白基因会对低浓度(10 ng·L^(-1)和20 ng·L^(-1))的Aroclor 1254短时间(1 d)暴露产生转录上调响应,且在成体阶段相关基因的转录响应更强烈;与成体阶段相比,文蛤幼体阶段暴露于高浓度(2000 ng·L^(-1))的Aroclor 1254中,生长与发育、蛋白质代谢与免疫调控过程相关的MmTR潜在互作蛋白基因RSPH9、N23、ferritin、BHMT、SCC4、PSMB5具�Thyroid disrupting chemicals(TDCs)are environmental endocrine disruptors,able to interfere with synthesis,secretion,transport,metabolism and action of thyroid hormones(THs),altering hormonal homeostasis and level in organisms.Thyroid hormone receptors(TRs)are DNA-binding transcription factor in the cell nucleus and are part of the ligand-dependent nuclear hormone receptor superfamily.Specific binding of the TRs DNA-binding domain to the thyroid hormone response elements(TREs)regulates gene expression.TDCs,acting as a receptor for TRs,competitively bind to TRs to regulate genes expression in thyroid hormone signaling pathway.To explore the interfering effects and mechanism of TDCs to the THs in marine invertebrates,the bait protein expression plasmid pGBKT7-MmTR and the cDNA library of digestive tissue from Meretrix meretrix were constructed.Results displayed that the length of the cDNA fragments were 0.5 to 2 kb,and the long-distributed smear indicated the successful synthesis of the ds cDNA.The titer of the primary cDNA library was above 2×10^(6) cfu.Meanwhile,it was verified that the non-toxic recombinant bait vector pGBKT7-MmTR has self-activation activity,and can be applied to the yeast two-hybrid system.From the yeast two hybrid system,a total of 36 positive clones were obtained through the screening of library,of which 30 clones had completely correct sequence.The positive result of random point-to-point validation of BIRC23 and MmTR genes concluded that all the presumptive results are reliable.After multiple screening with the high screening rate medium,rotary verification,sequencing and bioinformatics analysis,the interactions of the MmTR with 29 potential interacting proteins associated with functions in growth and development,protein metabolism and immune regulation,response to stress and transcriptional regulation.After the exposure to different concentrations of Aroclor 1254,the transcriptional response of MmTR and these 29 potential interacting proteins in M.meretrix during larvae and adult stages w

关 键 词：甲状腺干扰物 文蛤 酵母双杂交 互作蛋白 甲状腺激素受体 

分 类 号：X171.5[环境科学与工程—环境科学]