發(fā)布時間：2018-07-09 16:22

  本文選題：Spag6L基因 + 精子發(fā)生��； 參考：《武漢科技大學》2016年碩士論文

【摘要】：目的:構建小鼠Spag6L基因的真核表達質(zhì)粒,并研究其在哺乳動物細胞內(nèi)的表達與定位。構建上游啟動子熒光素酶報告基因重組質(zhì)粒,并檢測其轉錄活性。方法:以小鼠的睪丸c DNA基因文庫作為模版,PCR擴增Spag6L基因全長序列,測序正確后亞克隆至p EGFP-N2和pc DNA3真核表達載體中并酶切鑒定。將構建成功的重組表達質(zhì)粒轉染至COS-7與CHO細胞中,Western-blot法檢測COS-7細胞中Spag6L蛋白表達,激光共聚焦掃描顯微鏡觀察Spag6L蛋白在CHO細胞內(nèi)的定位。構建上游啟動子熒光素酶報告基因重組質(zhì)粒Spag6L/PGL3,用雙熒光素酶法檢測其轉錄活性并與Spag6進行比較。結果:重組質(zhì)粒Spag6L/pEGFP和Spag6L/pc DNA3經(jīng)雙酶切后產(chǎn)生的1.5kb的目的插入片段,經(jīng)測序證實與Spag6L基因一致。GFP-Spag6L融合蛋白分子量為82k D,Spag6L蛋白分子質(zhì)量為56k D。Spag6L蛋白在CHO細胞中主要定位于細胞質(zhì),以微管和囊泡表達為主。Spag6L上游啟動子轉錄調(diào)節(jié)序列熒光素酶活性明顯強于Spag6。結論:構建Spag6L/p EGFP、Spag6L/pc DNA3和Spag6L/PGL3重組質(zhì)粒成功,為進一步探究Spag6L基因與Spag6基因的關系以及Spag6L基因在精子發(fā)生中的作用奠定了基礎。
[Abstract]:Aim: to construct the eukaryotic expression plasmid of mouse Spag6L gene and study its expression and localization in mammalian cells. The recombinant plasmid of luciferase reporter gene of upstream promoter was constructed and its transcriptional activity was detected. Methods: the full-length sequence of Spag6L gene was amplified by PCR using mouse testis c DNA library as template, and subcloned into pEGFP-N2 and pcDNA3 eukaryotic expression vectors and identified by restriction endonuclease digestion. The recombinant expression plasmid was transfected into COS-7 and Cho cells to detect the expression of Spag6L protein by Western-blot. The localization of Spag6L protein in Cho cells was observed by confocal laser scanning microscopy. The upstream promoter luciferase reporter gene recombinant plasmid Spag6L / PGL3 was constructed and its transcriptional activity was detected by double luciferase method and compared with that of Spag6. Results: the recombinant plasmids Spag6L / pEGFP and Spag6L / pcDNA3 were inserted into the 1.5kb fragment. The molecular weight of the recombinant plasmid Spag6L / pEGFP and Spag6L / pcDNA3 was 56kD.Spag6L, which was consistent with that of Spag6L gene. The molecular weight of the fusion protein was 82kD / Spag6L, which was mainly located in the cytoplasm of Cho cells. The luciferase activity of transcriptional regulatory sequence of upstream promoter of microtubule and vesicle was significantly higher than that of Spag6L. Conclusion: the recombinant plasmids Spag6L / p EGFPN Spag6L / pcDNA3 and Spag6L / PGL3 were successfully constructed, which laid a foundation for further study of the relationship between Spag6L gene and Spag6 gene and the role of Spag6L gene in spermatogenesis.
【學位授予單位】：武漢科技大學
【學位級別】：碩士
【學位授予年份】：2016
【分類號】：R698.2

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