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MicroRNAs(miRNAs)通过抑制编码基因的转录调控关键的生物学进程。研究发现,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的发育过程中起着极其重要的作用。利用Solexa测序技术从牙鲆中成功鉴定出let-7家族的10个成员((let-7a,let-7b,let-7c,let-7d,let-7e,let-7f,let-7g,let-7h,let-7i,let-7j)。相对于let-7家族其他成员,let-7d的拷贝数最多,推测其在牙鲆发育过程中扮演着更重要的角色。运用实时荧光定量PCR方法分析let-7d在牙鲆变态发育不同时期的表达,结果发现,在对照组和甲状腺激素处理组,let-7d的表达模式类似:前5个时期(孵化后17 d,17dph;变态Ⅰ期,20 dph;变态Ⅱ期,23 dph;变态Ⅲ期,29 dph;变态Ⅳ期,36dph)表达量逐渐上升;变态晚期(36 dph)达到高峰;变态结束(41 dph)表达量呈现下降的趋势。定量结果显示,甲状腺激素下调let-7d的表达,与对照组呈显著性差异(P<0.05)。对牙鲆不同组织中let-7d的表达情况分析表明,let-7d主要存在于牙鲆的头部(脑,鳃,心脏)。靶基因预测发现,在目前已公布的482条牙鲆mRNA序列中,其中有30条mRNA的3’非翻译区(3’-UTRs)存在let-7d结合位点。这些预测的靶基因涉及到发育过程的许多方面:蛋白折叠,细胞分化,信号转导,病毒免疫反应和信号通路等。这些结果说明,let-7d在调控牙鲆从仔鱼向稚鱼转变的变态发育过程中起着极其重要的作用。
MicroRNAs (miRNAs) regulate the critical biological processes by inhibiting the transcription of the encoded genes. The study found that let-7 is an important regulatory factor in the development of timing and plays an extremely important role in promoting the development of larvae to adult. The let-7 family of 10 members (let-7a, let-7b, let-7c, let-7d, let-7e, let-7f, let-7g, let) was successfully identified from Japanese flounder using the Solexa sequencing technique Let-7i, let-7j). Compared with other members of let-7 family, let-7d has the most copy number, suggesting that it plays a more important role in the development of Japanese flounder.Using real-time fluorescence quantitative PCR The expression of let-7d in different stages of the Japanese flounder metamorphosis was analyzed. The expression pattern of let-7d was similar in the control group and the thyroid hormone treatment group. The expression pattern of let-7d was similar to that of the first five periods (17d, 17dph, (Dph, 20 dph; metamorphosis Ⅱ, 23 dph; metamorphosis Ⅲ, 29 dph; metamorphosis Ⅳ, 36dph) gradually increased; the peak of metamorphosis reached its peak at 36 dph; Quantitative analysis showed that the expression of let-7d in thyroid hormones was down-regulated significantly (P <0.05). The let-7d expression in different tissues of Japanese flounder showed that let-7d mainly existed in the flounder Head (brain, gill, heart) .According to the predicted target genes, we found that there are let-7d junctions in the 3’-untranslated region (3’-UTRs) of 30 mRNAs among the 482 flounder mRNA sequences published so far These predicted target genes involved in many aspects of the development process: protein folding, cell differentiation, signal transduction, viral immune response and signaling pathway, etc. These results show that let-7d in regulating flounder from larvae to naive Metamorphosis of fish changes play an extremely important role in the process.