RNAi干扰技术在生物节律相关基因hper1和clock研究中的应用

发布时间：2018-05-19 03:33

  本文选题：RNA干扰 + siRNA　； 参考：《四川大学》2007年硕士论文

【摘要】： RNA干扰(RNAi)是指内源性或外源性双链RNA通过一定的作用机制导致细胞内特定基因在mRNA水平上的表达显著降低。RNA干扰技术是过去十年里基因组学方面最引人注目的发现之一，该技术是应用21nt的短双链RNAs作为一个短干扰RNAs(siRNAs)当它介入到哺乳动物细胞内时有效的抑制RNA的表达。RNA干扰技术因其具有高度特异性和显著的干扰效果，而在研究和应用领域受到高度重视。 时间生物学的研究表明，人类的很多生命活动具有一定的节律。在各种不同周期的节律当中，近日节律是最引人注意，被研究最多的。Circadian，是一个最近几十年才出现的词。它被用来描述周期大概为24小时的节律。人类有很多节律活动的周期接近24小时。 近日节律(circadian rhythms)由生物钟(近日节律钟)系统产生和调控。该系统在调节生物的生理功能，并使之与外界环境相适应方面起着重要的作用。目前研究证明PER1是近日节律分子振荡中的关键性蛋白，主要通过蛋白与蛋白作用的方式发挥功能。但PER1通过何种途径发挥作用目前还不十分清楚。因此本文构建针对hper1的高效RNA干扰质粒，为进一步研究hper1的功能构建平台，并在此基础上对hper1的功能进行初步研究。结果RT-PCR鉴定显示pTER-hper1-Ⅱ干扰质粒干扰效率最高，hper1表达量降低84.9％。Western Blot显示hPER1表达受抑制后，，P—p44／42 MAPK水平升高。 在哺乳动物，节律基因per1，per2，Bmall，Clock和cry1已经在精子生成过程中被发现但却不是以近日节律的形式存在。值得注意的是Clock基因的表达仅仅在圆形精子的顶体被发现，这一发现推测Clock基因在雄性动物的生殖能力中可能起到了潜在的作用。因此在本研究中我们利用RNAi技术干扰clock在小鼠睾丸中的表达，结果westernblot显示在注射clock干扰质粒后3天和6天clock表达量分别下降55％和48％，为进一步探讨clock对小鼠生殖功能的影响构建平台，并在此基础上对clock对小鼠生殖功能影响进行初步研究，
[Abstract]:RNA interference (RNAi) is one of the most remarkable genomics discoveries in the past decade, which means that endogenous or exogenous double-stranded RNA can significantly reduce the expression of specific genes at the mRNA level through a certain mechanism. This technique uses 21nt's short double-stranded RNAs as a short interfering RNAssiRNAs. when it is involved in mammalian cells, it effectively inhibits the expression of RNA. RNAi has a highly specific and significant interference effect. And in the field of research and application is highly valued. Studies in chronobiology have shown that many human life activities have a certain rhythm. Of all the rhythms in different cycles, circadian rhythm is the most noticeable and studied. It is a word that has only appeared in recent decades. It is used to describe the rhythm of a cycle of about 24 hours. Humans have many rhythmic activities with cycles approaching 24 hours. Circadian rhythms are produced and regulated by the circadian rhythms system. The system plays an important role in regulating biological physiological function and adapting it to the external environment. It has been proved that PER1 is the key protein in circadian molecular oscillations and functions mainly through the interaction of protein and protein. But the way in which PER1 works is unclear. In order to further study the function of hper1, this paper constructs an efficient RNA interference plasmid for hper1, and makes a preliminary study on the function of hper1. Results RT-PCR analysis showed that pTER-hper1- 鈪

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