牙鲆let-7基因的克隆与表达及其靶基因预测  

Cloning,expression and target gene prediction of let-7 in Paralichthys olivaceus metamorphosis

作　　者：施志仪[1] 吴明林[1] 付元帅[1] 

机构地区：[1]上海海洋大学水产与生命学院,上海201306

出　　处：《中国水产科学》2011年第6期1211-1218,共8页Journal of Fishery Sciences of China

基　　金：国家自然科学基金项目(31172392);上海重点学科项目(S30701)

摘　　要：let-7是时序发育过程中的重要调控因子,在促进幼体向成体转变过程中起着极其重要的作用。为探讨let-7对牙鲆(Paralichthys olivaceus)从幼体到成体转变的变态发育过程的影响,本实验对let-7进行了克隆与表达研究,并预测了其可能的靶基因。利用普通PCR技术从牙鲆中成功鉴定出let-7及其前体序列,该前体序列在不同的物种间具有高度的保守性。运用实时荧光定量RT-PCR方法分析let-7在牙鲆变态发育不同阶段的表达,结果发现,水温(16±0.5)℃条件下,在牙鲆变态发育的前5个阶段(孵化后17、20、23、29、36 d),let-7的表达量逐渐上升,在变态末期(36 dph)达到高峰,变态结束(41 dph)时表达量呈现下降的趋势。实时荧光定量数据表明,甲状腺激素(T3)上调牙鲆肾细胞中let-7的表达,在浓度为75 nmol/L时,表达量达到最高。靶基因预测发现,let-7作用于多样的靶基因,其靶基因可能参与生长发育、细胞分化、信号转导、病毒免疫等生物过程。本研究建立了let-7在牙鲆变态发育过程中的表达谱,并在细胞水平研究了T3对let-7表达量的影响,预测了其可能的靶基因,为研究let-7在牙鲆变态发育过程中的调控机制奠定了基础。The let-7 gene is a critical regulator of developmental timing events of the larval-to-adult transition. Here, Paralichthys olivaceus let-7 was identified by a stem-loop RT-PCR. The precursor of let-7 was cloned and was proven to be highly conserved among different species. Quantitative analysis revealed that the expression levels of let-7 were generally variably enhanced during the first five metamorphic stages and peaked during the late metamorphic stages （36 dph）. At the end of P. olivaceus metamorphosis （41 dph）, expression of let-7 was reduced. To study the effects of triiodothyronine （T3） on the expression of let-7, six different concentrations （0 nmol·L^-1, 50 nmol·L^-1, 75 nmol·L^-1, 100 nmol·L^-1, 200 nmol·L^-1） were used to treat P. olivaceus kidney cells in vitro. Quantitative PCR showed that let-7 levels increased after T3 treatment, especially at 75 nmol·L^-1, indicating that T3 regulated the expression of let-7. Further investigation of target genes demonstrated that the 3′-untranslated regions （3￠-UTRs） of 29 mRNAs were possible target sites matched with let-7. These mRNA targets played key roles in diverse biological processes： development, cell proliferation, signal transduction, virus immune response, and signaling pathways. These re sults suggested that let-7 played an essential role in regulating the developmental timing of P. olivaceus metamorphosis.

关 键 词：牙鲆 LET-7 克隆与表达 变态 甲状腺激素 靶基因 

分 类 号：S91[农业科学—水产科学]