当前位置: 首页 > 文章 > 牙鲆let-7基因的克隆与表达及其靶基因预测 中国水产科学 2011,18 (6) 1211-1218
Position: Home > Articles > Cloning,expression and target gene prediction of let-7 in Paralichthys olivaceus metamorphosis Journal of Fishery Sciences of China 2011,18 (6) 1211-1218

牙鲆let-7基因的克隆与表达及其靶基因预测

作  者:
施志仪;吴明林;付元帅
单  位:
上海海洋大学水产与生命学院
关键词:
牙鲆;let-7;克隆与表达;变态;甲状腺激素;靶基因
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。
译  名:
Cloning,expression and target gene prediction of let-7 in Paralichthys olivaceus metamorphosis
作  者:
SHI Zhiyi,WU Minglin,FU Yuanshuai College of Fisheries and Life Science,Shanghai Ocean University,Shanghai 201306,China
关键词:
Paralichthys olivaceus;let-7;cloning and expression;metamorphosis;triiodothyronine(T3);target gene
摘  要:
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 re-duced.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.

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