本文選題：真核翻譯起始因子　切入點(diǎn)：真核翻譯延伸因子　出處：《塔里木大學(xué)》2017年碩士論文

【摘要】：羊毛的生長(zhǎng)發(fā)育受多種因素的影響,其生長(zhǎng)發(fā)育過(guò)程和周期性生理調(diào)控對(duì)羊毛性狀相關(guān)基因的測(cè)定及調(diào)控網(wǎng)絡(luò)的探索有著積極的影響。真核翻譯起始因子3L(EIF3L)、真核翻譯起始因子3H(EIF3H)、真核翻譯延伸因子(EEF1D)是一類在細(xì)胞內(nèi)參與調(diào)控蛋白質(zhì)翻譯的細(xì)胞因子,與細(xì)胞活動(dòng)緊密相關(guān)。本研究首先以中國(guó)美利奴羊成纖維細(xì)胞總RNA為模板,用RT-PCR擴(kuò)增獲得EIF3L、EIF3H、EEF1D基因mRNA的CDS區(qū)編碼序列,通過(guò)酶切及純化后,分別連接至原核表達(dá)載體pet28α(+)、真核表達(dá)載體pcDNA3.1(-),構(gòu)建EIF3L、EIF3H、EEF1D基因的原核表達(dá)載體和真核表達(dá)載體。測(cè)序結(jié)果顯示,獲得的目的基因序列與Genebank上綿羊預(yù)測(cè)序列相似性分別為100%、99.91%、100%,其中EIF3H序列第1055bp位置上的核苷酸堿基由A突變?yōu)镚,編碼的氨基酸由天冬酰胺(Asn/N)突變?yōu)锳GC絲氨酸(Ser/S);對(duì)序列比對(duì)分析發(fā)現(xiàn),EIF3L、EIF3H、EEF1D核苷酸序列及編碼的氨基酸與人、牛、鼠、豬、兔等動(dòng)物的核苷酸序列及編碼的氨基酸序列有一定的同源性。將重組的原核表達(dá)質(zhì)粒轉(zhuǎn)入E.coli BL21(DE3)后,在37℃,終濃度為0.75mmol·L~(-1)的IPTG誘導(dǎo)3.5 h后,分別產(chǎn)生了以包涵體形式表達(dá)的蛋白,其中EIF3L蛋白分子量大小約為69kDa,EIF3H蛋白分子量大小約為40kDa,EEF1D蛋白分子量大小約為40kDa,經(jīng)Western blot檢測(cè)該蛋白分子具有很好的反應(yīng)原性。將構(gòu)建好的重組真核表達(dá)載體分別瞬時(shí)轉(zhuǎn)染至原代培養(yǎng)的中國(guó)美利奴羊成纖維中,再將轉(zhuǎn)染細(xì)胞分別提取總RNA和總蛋白,進(jìn)行RT-qPCR和Western blot檢測(cè)后,結(jié)果顯示,EIF3L、EIF3H的mRNA表達(dá)量和蛋白表達(dá)量相對(duì)于正常的成纖維細(xì)胞均有不同程度的上調(diào),EEF1D的mRNA表達(dá)量未出現(xiàn)明顯上調(diào),蛋白表達(dá)量升高。在與羊毛相關(guān)基因表達(dá)量檢測(cè)中發(fā)現(xiàn),轉(zhuǎn)染重組真核表達(dá)載體的成纖維細(xì)胞mRNA表達(dá)量分別有著不同程度的上調(diào)和下調(diào)。采用RT-qPCR和Western blot對(duì)中國(guó)美利奴羊和哈薩克羊進(jìn)行組織表達(dá)譜檢測(cè),結(jié)果顯示,EIF3L、EIF3H、EEF1D的mRNA和蛋白在中國(guó)美利奴羊和哈薩克羊的心臟、肝臟、脾臟、肺臟、腎臟、皮膚、肌肉組織中均有不同程度的表達(dá)且具有一定差異。
[Abstract]:The growth and development of wool are affected by many factors. Its growth and development process and periodic physiological regulation have a positive effect on the determination of the genes related to wool traits and the exploration of regulatory networks. The eukaryotic translation initiation factor 3L, the eukaryotic translation initiation factor 3H, the eukaryotic translation initiation factor 3H, the eukaryotic translation extension factor EEF1D). Is a class of cytokines involved in the regulation of protein translation in cells. In this study, the total RNA of Chinese Merino sheep fibroblasts was used as a template to amplify the coding sequence of the CDS region of the EIF3L- EIF3HIF3HEEEF1D gene mRNA, which was digested and purified by enzyme digestion. The prokaryotic expression vector and eukaryotic expression vector of EIF3L- EIF3HEEF1D gene were constructed by ligating to the prokaryotic expression vector pet28 偽 (pet28 偽) and eukaryotic expression vector pcDNA3.1HEEF1D, respectively. The similarity between the obtained target gene sequence and the predicted sequence of sheep on Genebank is 100 ~ 99.91%, in which the nucleotide base at the 1055bp position of the EIF3H sequence is mutated from A to G, and the encoded amino acid is mutated from asparagine Asn / N to AGC serine serine serine. The nucleotide sequence and amino acid encoding of EIF3L, EIF3H, EEF1D and human were found by comparison analysis. The nucleotide sequence and amino acid sequence of bovine, mouse, pig, rabbit and so on have some homology. After the recombinant prokaryotic expression plasmid was transferred into E.coli BL21 (DE3), the recombinant plasmid was induced by IPTG at 37 鈩，

本文編號(hào)：1676097