The role of non-coding RNAin developmental toxicity by dioxin

1)以转录组为靶点的新型DOP-PCR方法的建立：我们建立了一种重复性较高的简并寡核苷酸启动的新方法(DOP-PCR)。由于斑马鱼(Danio Rerio)转录组中存在多套适合进行聚合酶链式反应的反向重复序列，因此我们采用了单引物法进行DOP-PCR。使用我们开发的计算机程序，设计了引物的引物部分(特定序列部分)，为每个PCR反应获得约10条条带。2)差异显示方法寻找TCDD增加的转录：我们进行了基于上述DOP-PCR的差异显示，发现TCDD暴露显著上调了CYP1C1和1C2。CYP1C2基因敲除可挽救TCDD诱导的中脑循环衰竭，提示其在TCDD发育毒性中的可能规律。3)斑马鱼引物DOP-PCR在其他鱼类中的应用：针对斑马鱼设计的引物的DOP-PCR可用于青海等8个鱼种的可重复的PCR产物。4)二恶英对miRNA转录状态的影响：采用含有272个不同miRNA斑点的微阵列培养基来阐明TCDD对miRNA转录状态的影响。在受精后48小时和72小时，约有10种miRNA物种在TCDD暴露下的受精水平显示出两倍或更多的变化。这些miRNA大多是在两个阶段之间重叠的。

1) Development of novel DOP-PCR method with transcriptome as a target: We developed a novel degenerate oligonucleotide-primed PCR (DOP-PCR) with high reproducibility. We used mono-primers for DOP-PCR, as there were many sets of inverse-repeat sequence adequate for PCR reaction in transcriptome of zebrafish (Danio rerio). Primed part of primers (specific sequence part) was designed to obtain about 10 bands for each PCR reaction, using our developed computer program.2) Differential display approach in search for transcripst increased by TCDD : We carried out differential display based on DOP-PCR described above and found CYP1C1 and 1C2 were dramatically upregulated by TCDD exposure. Gene knockdown of CYP1C2 rescued mesencephalic circulation from TCDD-induced failure, suggesting the possible rule of CYP1C2 in TCDD developmental toxicity.3) Application of DOP-PCR for other fish species with zebrafish primers: DOP-PCR with the primers designed for zebrafish could be used for reproducible PCR products with medaka (Oryzias latips) and other 8 fish species. The same strategy could be applicable for various mammals or birds using DOP-PCR primers designed for rodents and domestic fowls.4) Effects of dioxin on miRNA transcriptional status: Microarray media with 272 different miRNA spots was used to clarify the effect of TCDD on miRNA transcriptinal status. About 10 miRNA species showed two-fold changes or more in trasciptational level by TCDD exposure at 48 and 72 hours post fertilization, respectively. Most of these miRNA speceis overlap between two stages.