非编码RNA讲课.ppt

5、/PMRD/ 5、/PMRD/ 5、/PMRD/ 6、/pmirkb/ 6、/pmirkb/ 6、/pmirkb/ 6、/pmirkb/ 6、/pmirkb/ 6、/pmirkb/ miRNA与靶基因功能研究 1、确定miRNA与靶基因为互作关系 （1）软件预测（前面介绍的多个网站） （2）small RNA测序 （3）降解组测序 （4）用RACE(rapid-amplification of cDNA ends)方法确定切点 （5）用miRNA作用的靶位点修饰或者不修饰的mRNA过表达，分析mRNA表达水平与表型的变化。 （6）过表达miRNA增加miRNA表达，或者用target mimicry降低miRNA的表达，分析靶mRNA的变化。 miRNA与靶基因功能研究 （3）降解组测序可以确定miRNA对靶mRNA的切点 miRNA与靶基因功能研究 （4）用RACE(rapid-amplification of cDNA ends) 确定切点的常用方法是： RNA ligase mediated amplification of cDNA ends（RLM-RACE） miRNA与靶基因功能研究 （5）用miRNA作用的靶位点修饰 miRNA与靶基因功能研究 （5）用miRNA作用的靶位点修饰 modified target： 通过替换密码子的第三个碱基，只改变核酸序列，不改变氨基酸序列 miRNA与靶基因功能研究 （6）target mimicry原理 Target mimicry provides a new mechanism for regulation of microRNA activity. Nat Genet. 2007, 39(8):1033-7. WT miRNA miRNA miRNA过表达 miRNA靶位点修饰 miRNA靶位点未修饰 扎实基础、宽广视野， 大胆假设、认真求证， 坚定信念、勤奋工作。 Analysis of the genome sequence of the flowering plant Arabidopsis thaliana. Nature. 2000，408(6814):796-815. gene density of one gene per 4.5Kb The map-based sequence of the rice genome. Nature. 2005, 436(7052):793-800. 1、provide evidence for a genome size of 389 Mb. This size estimation is approx260 Mb larger than the fully sequenced dicot plant model Arabidopsis thaliana. We generated 370 Mb of finished sequence, representing 95% coverage of the genome and virtually all of the euchromatic regions. 2、A total of 37,544 non-transposable-element-related protein-coding sequences were detected, compared with approx28,000–29,000 in Arabidopsis, with a lower gene density of one gene per 9.9 kb in rice. The sequence of the human genome. Science. 2001，16;291(5507):1304-51. Size of the genome (including gaps) 2.91 GB Percent of genome classified as repeats 35 Number of annotated genes 26,383 Percent of annotated genes with unknown function 42 Number of genes (hypothetical and annotated) 39,114 Percent of hypothetical and annotated genes with unknown function 59 Average gene size 27 kb 从基因组测序结果显示： 基因组 上编码蛋白质的区域（也就是通常说的基因）只占人类基因组的一小部分，不会超过整个基因组的3%, 其余97%