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Alternative TitleStudy of Short Interfering RNA (siRNA) Produced in Transcription System of Zebrafish Embryo
Thesis Advisor朱作言 ; 孙永华
Degree Grantor中国科学院水生生物研究所
Place of Conferral水生生物研究所
KeywordRna干扰 斑马鱼 H1启动子 U6启动子 T7 Rna聚合酶 T7启动子 Shrna载体
AbstractRNA干扰是1998年在线虫中发现的一种转录后基因沉默现象,是指由双链RNA引发的同源mRNA表达受阻或降低的过程。目前利用RNA干扰进行基因功能,基因调控,基因治疗等方面的研究已经在植物,无脊椎动物,哺乳动物细胞中广泛开展起来。然而,作为一种越来越受人们关注的模式生物,斑马鱼在RNAi应用方面的研究远远没有达到像在低等生物,小鼠中的程度,还亟需深入探讨。迄今为止,关于斑马鱼中RNAi研究的报道为数不多,而且都没有得到一致的结论,甚至相互矛盾。那么RNAi到底能否在斑马鱼中发挥抑制基因功能的作用呢?本研究首次克隆斑马鱼自身H1,U6启动子,分析其转录活性后构建了分别由这两个启动子驱动的shRNA载体,通过在鲤鱼细胞和斑马鱼胚胎中的分析发现此载体可以部分地抑制内外源基因的表达;首次利用噬菌体T7 RNA聚合酶/T7启动子系统在斑马鱼中进行RNAi的研究,结果证实该系统可以在斑马鱼胚胎中正常工作,而且构建的由T7启动子驱动的shRNA载体可以在表达T7 RNA聚合酶的斑马鱼品系中有效的抑制内外源基因的表达。本研究为RNAi在斑马鱼中的应用开辟了一个很好的研究基础,为更好的研究基因功能提供了广阔的前景。 关键词:RNA干扰;斑马鱼;H1启动子;U6启动子;T7 RNA聚合酶;T7启动子;shRNA载体
Other AbstractRNA interference (RNAi) is the phenomenon of post-transcriptional gene silencing (PTGS) found in C. elegans in 1998 in which dsRNA specifically blocks the expression of its homologous gene. RNAi has been widely applied in many organisms such as plants, lower animals and higher vertebrates as a tool to study gene function, gene regulation and gene therapy. However, as a model organism receiving more and more attention, the study of RNAi application in zebrafish has not developed with convenient technology as that in the lower animals and mouse. So far, reports on the study of RNAi in zebrafish have not reached the consistent conclusions, but even controversial conclusions. Thus, it remains a question that whether RNAi works in zebrafish as a tool to study gene function. In this project we firstly described the cloning, transcription activity analysis and RNAi application of zebrafish H1 and U6 promoters. By ways of experiments in cultured fish cells and zebrafish embryos, shRNA vectors driven by zebrafish H1 and U6 promoters could knock down the transcription levels of foreign and endogenous genes. This study also applied bacteriophage T7 RNA polymerase/T7 promoter system in RNAi study of zebrafish embryos and the results confirmed that this system could work well in zebrafish embryos. Furthermore, shRNA vector driven by T7 promoter inhibited effectively the target genes expression in transgenic zebrafish embryos which expressed stably T7 RNA polymerase. This study provided a good foundation for further application of RNAi in zebrafish and a wide profoundness for study gene function in zebrafish by ways of RNAi. Keywords: RNA interference, zebrafish, H1 promoter, U6 promoter, T7 RNA polymerase, T7 promoter, shRNA vector
Document Type学位论文
Recommended Citation
GB/T 7714
王娜. 利用斑马鱼胚胎转录系统产生小分子干扰RNA(siRNA)的研究[D]. 水生生物研究所. 中国科学院水生生物研究所,2007.
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