發(fā)布時(shí)間：2018-03-22 18:08

  本文選題：綿羊　切入點(diǎn)：胎兒　出處：《中國農(nóng)業(yè)大學(xué)》2016年博士論文　論文類型：學(xué)位論文

【摘要】：LncRNA是一類長度大于200個(gè)nt且不表現(xiàn)出蛋白質(zhì)編碼潛能的RNAs。參與了X染色體沉默,基因組印記以及染色質(zhì)修飾,轉(zhuǎn)錄激活,轉(zhuǎn)錄干擾,核內(nèi)運(yùn)輸?shù)榷喾N重要的調(diào)控過程,涉及到表觀遺傳調(diào)控、轉(zhuǎn)錄調(diào)控及轉(zhuǎn)錄后調(diào)控等多個(gè)層面。最新報(bào)道LncRNA分子可以調(diào)節(jié)人和小鼠的肌肉功能,但LncRNA對(duì)肉用家畜肌肉分化發(fā)育調(diào)控的研究尚無報(bào)道。本研究通過高通量測(cè)序的手段篩選到一個(gè)的可以促進(jìn)綿羊肌肉細(xì)胞分化,促進(jìn)成肌生成的LncRNA分子(命名為Lnc-SEMT),試驗(yàn)分析發(fā)現(xiàn)它對(duì)骨髂肌發(fā)育具有重要調(diào)控作用.該分子的研究為闡明動(dòng)物肌細(xì)胞的增殖、分化的分子機(jī)制具有重要意義,并可為肉羊分子輔助育種和開發(fā)新型的促生長制劑提供科學(xué)依據(jù)和技術(shù)支持。主要研究結(jié)果如下：1.通過對(duì)胚胎期90口和120日齡綿羊背最長肌樣品進(jìn)行高通量測(cè)序,共計(jì)得到1114個(gè)lncRNA,其中466個(gè)lncRNA差異表達(dá),而466個(gè)差異表達(dá)的lncRNA中,其中157個(gè)lncRNA上調(diào),309個(gè)lncRNA下調(diào)。2.通過對(duì)測(cè)序得到的1114個(gè)lncRNA進(jìn)行芯片驗(yàn)證,發(fā)現(xiàn)有867個(gè)預(yù)測(cè)的lncRNA存在,其中有119個(gè)lncRNA的表達(dá)上調(diào),對(duì)這119個(gè)lncRNA進(jìn)行定量篩選后,得到了一個(gè)在骨骼肌組織中特異性高表達(dá)的上調(diào)LncRNA(命名為Lnc-SEMT)。3.通過檢測(cè)綿羊不同生長時(shí)期的Lnc-SEMT表達(dá)水平發(fā)現(xiàn),在胚胎期120日齡時(shí),其表達(dá)量最高,出生后逐漸降低,成年后表達(dá)水平變化不大。4.通過檢測(cè)綿羊成肌細(xì)胞生長分化過程中的lncRNA表達(dá)量變化,在成肌細(xì)胞分化0-5天內(nèi),隨著成肌細(xì)胞的生長分化,MYOG和MYOD基因表達(dá)水平也隨著升高。5.構(gòu)建了Lnc-SEMT的高表達(dá)和干擾載體,分別轉(zhuǎn)染到綿羊成肌細(xì)胞,通過DAPI和MyHC試驗(yàn)分析,結(jié)果表明隨著成肌細(xì)胞分化過程中Lnc-SEMT的過表達(dá)和干擾,MYOG, MYOD等基因隨著Lnc-SEMT的變化而變化。6.通過顯微注射的手段,得到了5只轉(zhuǎn)Lnc-SEMT綿羊。轉(zhuǎn)基因突變綿羊的Lnc-SEMT表達(dá)水平是野生型綿羊的3.3倍。7.通過對(duì)轉(zhuǎn)Lnc-SEM T綿羊出生后的體重進(jìn)行測(cè)量,發(fā)現(xiàn)突變羊在3月齡,6月齡和9月齡的體重都顯著高于對(duì)照野生組。另外屠宰試驗(yàn)中突變羊的胴體重也顯著高于對(duì)照野生組。8.通過對(duì)轉(zhuǎn)Lnc-SEMT綿羊進(jìn)行HE染色和免疫熒光染色,發(fā)現(xiàn)突變羊的肌纖維的橫截面積顯著高于對(duì)照組,另外,突變羊的30-45μm直徑的肌纖維顯著高于對(duì)照組,其他直徑的肌纖維差異不明顯。9.通過應(yīng)用軟件,預(yù)測(cè)到了Lnc-SEMT 與 miR-125 b的潛在結(jié)合位點(diǎn)。10.對(duì)Lnc-SEMT 于 miR-125b的結(jié)合位點(diǎn)進(jìn)行突變,并進(jìn)行體外轉(zhuǎn)染試驗(yàn),發(fā)現(xiàn)miR-125b確實(shí)存在與L nc-SEMT潛在的結(jié)合位點(diǎn),同時(shí)Lnc-SEMT與miR-125b競(jìng)爭(zhēng)性的結(jié)合IGF-2,以實(shí)現(xiàn)促進(jìn)成肌細(xì)胞分化和成肌生成的作用。11.通過對(duì)轉(zhuǎn)Lnc-SEMT綿羊的血清進(jìn)行檢測(cè),發(fā)現(xiàn)轉(zhuǎn)Lnc-SEMT綿羊血液中的IGF-2表達(dá)量顯著高于對(duì)照野生組。
[Abstract]:LncRNA is a kind of length greater than 200 nt and does not show the protein encoding potential RNAs. in chromosome X silencing, genomic imprinting and chromatin modification, transcriptional activation, transcription regulation of nuclear interference, transportation and other important, involved in epigenetic regulation, transcriptional regulation and post transcriptional regulation of multiple the latest reported level. LncRNA molecules can regulate human and mouse muscle function, but there is no report on LncRNA development regulation of meat animal muscle differentiation. This study screened a can promote sheep muscle cell differentiation by means of high-throughput sequencing, promote LncRNA molecules into muscle formation (named Lnc-SEMT), test the analysis found that it has an important role on the development of skeletal muscle. The molecular research to elucidate animal muscle cell proliferation plays an important role in the molecular mechanism of differentiation, and for molecular assisted sheep 鑲茬鍜屽紑鍙戞柊鍨嬬殑淇冪敓闀垮埗鍓傛彁渚涚瀛︿緷鎹拰鎶，

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