本文選題：RNA干擾 + siRNA　； 參考：《四川大學(xué)》2007年碩士論文

【摘要】： RNA干擾(RNAi)是指內(nèi)源性或外源性雙鏈RNA通過一定的作用機(jī)制導(dǎo)致細(xì)胞內(nèi)特定基因在mRNA水平上的表達(dá)顯著降低。RNA干擾技術(shù)是過去十年里基因組學(xué)方面最引人注目的發(fā)現(xiàn)之一，該技術(shù)是應(yīng)用21nt的短雙鏈RNAs作為一個短干擾RNAs(siRNAs)當(dāng)它介入到哺乳動物細(xì)胞內(nèi)時有效的抑制RNA的表達(dá)。RNA干擾技術(shù)因其具有高度特異性和顯著的干擾效果，而在研究和應(yīng)用領(lǐng)域受到高度重視。 時間生物學(xué)的研究表明，人類的很多生命活動具有一定的節(jié)律。在各種不同周期的節(jié)律當(dāng)中，近日節(jié)律是最引人注意，被研究最多的。Circadian，是一個最近幾十年才出現(xiàn)的詞。它被用來描述周期大概為24小時的節(jié)律。人類有很多節(jié)律活動的周期接近24小時。 近日節(jié)律(circadian rhythms)由生物鐘(近日節(jié)律鐘)系統(tǒng)產(chǎn)生和調(diào)控。該系統(tǒng)在調(diào)節(jié)生物的生理功能，并使之與外界環(huán)境相適應(yīng)方面起著重要的作用。目前研究證明PER1是近日節(jié)律分子振蕩中的關(guān)鍵性蛋白，主要通過蛋白與蛋白作用的方式發(fā)揮功能。但PER1通過何種途徑發(fā)揮作用目前還不十分清楚。因此本文構(gòu)建針對hper1的高效RNA干擾質(zhì)粒，為進(jìn)一步研究hper1的功能構(gòu)建平臺，并在此基礎(chǔ)上對hper1的功能進(jìn)行初步研究。結(jié)果RT-PCR鑒定顯示pTER-hper1-Ⅱ干擾質(zhì)粒干擾效率最高，hper1表達(dá)量降低84.9％。Western Blot顯示hPER1表達(dá)受抑制后，，P—p44／42 MAPK水平升高。 在哺乳動物，節(jié)律基因per1，per2，Bmall，Clock和cry1已經(jīng)在精子生成過程中被發(fā)現(xiàn)但卻不是以近日節(jié)律的形式存在。值得注意的是Clock基因的表達(dá)僅僅在圓形精子的頂體被發(fā)現(xiàn)，這一發(fā)現(xiàn)推測Clock基因在雄性動物的生殖能力中可能起到了潛在的作用。因此在本研究中我們利用RNAi技術(shù)干擾clock在小鼠睪丸中的表達(dá)，結(jié)果westernblot顯示在注射clock干擾質(zhì)粒后3天和6天clock表達(dá)量分別下降55％和48％，為進(jìn)一步探討clock對小鼠生殖功能的影響構(gòu)建平臺，并在此基礎(chǔ)上對clock對小鼠生殖功能影響進(jìn)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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