目的研究非编码小RNA-1896（miRNA-1896）和miRNA-409—3p对细胞因子信号抑制蛋白-3（suppressors of cytokinesignaling-3，SOTS3）基因的调控作用，构建floes3基因3’端非翻译区（3’-untranslated re-gion，3’-UTR）野生型和突变型...目的研究非编码小RNA-1896（miRNA-1896）和miRNA-409—3p对细胞因子信号抑制蛋白-3（suppressors of cytokinesignaling-3，SOTS3）基因的调控作用，构建floes3基因3’端非翻译区（3’-untranslated re-gion，3’-UTR）野生型和突变型重组荧光素酶报告载体。方法以原代培养的小鼠星形胶质细胞总eDNA为模板，通过点突变和缺失突变的方式分别对SOCS33’-UTR序列中miRNA-1896和miRNA-409—3p种子区的结合位点CAGAGA（897—902位）和AACATT（1425—1430位）进行突变，并将野生型的3’-UTR序列与突变的3’-UTR序列分别插入到虫荧光素酶表达质粒pGL3-Promoter获得重组质粒，命名为pGL3-SOCS3-WT、pGL3-SOCS3-M1和pGL3-SOCS3-M2。将上述重组质粒分别与miRNA-1896和miRNA-409—3p共转染至HEK293T细胞中，测定虫荧光素酶的活性。结果酶切验证及测序结果表明，重组质粒pGL3-SOCS3-WT、pGL3-SOCS3-M1和pGL3-SOCS3-M2构建成功。与对照组相比，转染miRNA-1896和miRNA-409—3p均显著降低了pGL3-SOCS3-WT虫荧光素酶的活性，但对pGL3-SOCS3-M1和pGL3-SOCS3-M2荧光素酶的活性并无明显影响。结论成功构建了soc33’-UTR野生型及突变型的虫荧光素酶重组表达质粒，确认CA-GAGA（897—902位）和AACATr（1425—1430位）分别是miRNA-1896和miRNA-409—3p种子区与socs33’-UTR结合的关键位点。展开更多
Pea ( Pisum sativum Linn.) actin gene family contains at least three isoforms (PEAcⅠ, PEAcⅡand PEAcⅢ), and the DNA sequence of these isoforms show high similarity in the coding regions and significant divergence...Pea ( Pisum sativum Linn.) actin gene family contains at least three isoforms (PEAcⅠ, PEAcⅡand PEAcⅢ), and the DNA sequence of these isoforms show high similarity in the coding regions and significant divergence in the untranslated regions. RT_PCR and Southern blotting using 3′_untranslated region (3′_UTR) as specific probe revealed that pea isoactin genes were expressed in roots, stems, leaves, tendrils, pollen and juvenile siliques, but displayed different patterns of transcript accumulation. Two_fold serial dilution electrophoresis showed PEAcⅠ mRNA preferentially accumulated in rapidly developing tissues: it peaked in seven days' stem; remained at a rather high level in leaves within a month but decreased significantly later; varied a little in tendrils and reached a median and a very low level respectively in juvenile siliques and in pollen. PEAcⅡ displayed somewhat similar expression pattern to PEAcⅠ. The observed differences in sequences and transcript accumulation patterns suggest that the individual pea actin genes may differ in their transcriptional regulation and cellular function. Phylogenetic tree of actins showed that pea actin isoforms are as diverged from each other as they are from other plant actins, and pea actins might have originated from a common ancestor before the divergence of the dicot and monocot plants.展开更多
利用噬菌体衣壳蛋白MS2和带有序列特异性茎环结构(含有MS2蛋白结合位点)的RNA之间的高度亲和力,对外源性人血管紧张素1型受体(angiotensin II receptor type 1,AGTR1)mRNA 3′端非翻译区(3′untranslated region,3′UTR)片段进行红色荧...利用噬菌体衣壳蛋白MS2和带有序列特异性茎环结构(含有MS2蛋白结合位点)的RNA之间的高度亲和力,对外源性人血管紧张素1型受体(angiotensin II receptor type 1,AGTR1)mRNA 3′端非翻译区(3′untranslated region,3′UTR)片段进行红色荧光标记,进而在活细胞(HeLa)内研究该mRNA片段的应激生物学行为。通过在pSG5空载体质粒上先后插入两个双链DNA目的片段AGTR1-3′UTR和24×MS2,构建重组质粒pSG5/AGTR1-3′UTR/24×MS2,并将该质粒与重组质粒pERFP/MS2和pEGFP/C1-G3BP共转染入Hela细胞。荧光显微成像结果显示,AGTR1-3′UTR-24×MS2 mRNA片段能够携带具有入核信号的MS2-RFP融合蛋白离开胞核进入胞浆,而且在亚砷酸盐刺激下,红色荧光标记的AGTR1-3′UTR-24×MS2 mRNA片段可在胞浆中形成与应激蛋白G3BP-GFP共定位的颗粒。该结果表明,针对AGTR1-3′UTR片段的MS2-RFP荧光标记系统构建成功,该荧光标记系统能有效避免假阳性的荧光信号。在细胞受到氧化应激时,AGTR1-3′UTR会被招募至胞浆中的应激颗粒结构中,启示了AGTR1-3′UTR区域对于调控AGTR1 mRNA在细胞内的应激定位具有重要作用。展开更多
文摘目的研究非编码小RNA-1896（miRNA-1896）和miRNA-409—3p对细胞因子信号抑制蛋白-3（suppressors of cytokinesignaling-3，SOTS3）基因的调控作用，构建floes3基因3’端非翻译区（3’-untranslated re-gion，3’-UTR）野生型和突变型重组荧光素酶报告载体。方法以原代培养的小鼠星形胶质细胞总eDNA为模板，通过点突变和缺失突变的方式分别对SOCS33’-UTR序列中miRNA-1896和miRNA-409—3p种子区的结合位点CAGAGA（897—902位）和AACATT（1425—1430位）进行突变，并将野生型的3’-UTR序列与突变的3’-UTR序列分别插入到虫荧光素酶表达质粒pGL3-Promoter获得重组质粒，命名为pGL3-SOCS3-WT、pGL3-SOCS3-M1和pGL3-SOCS3-M2。将上述重组质粒分别与miRNA-1896和miRNA-409—3p共转染至HEK293T细胞中，测定虫荧光素酶的活性。结果酶切验证及测序结果表明，重组质粒pGL3-SOCS3-WT、pGL3-SOCS3-M1和pGL3-SOCS3-M2构建成功。与对照组相比，转染miRNA-1896和miRNA-409—3p均显著降低了pGL3-SOCS3-WT虫荧光素酶的活性，但对pGL3-SOCS3-M1和pGL3-SOCS3-M2荧光素酶的活性并无明显影响。结论成功构建了soc33’-UTR野生型及突变型的虫荧光素酶重组表达质粒，确认CA-GAGA（897—902位）和AACATr（1425—1430位）分别是miRNA-1896和miRNA-409—3p种子区与socs33’-UTR结合的关键位点。
文摘Pea ( Pisum sativum Linn.) actin gene family contains at least three isoforms (PEAcⅠ, PEAcⅡand PEAcⅢ), and the DNA sequence of these isoforms show high similarity in the coding regions and significant divergence in the untranslated regions. RT_PCR and Southern blotting using 3′_untranslated region (3′_UTR) as specific probe revealed that pea isoactin genes were expressed in roots, stems, leaves, tendrils, pollen and juvenile siliques, but displayed different patterns of transcript accumulation. Two_fold serial dilution electrophoresis showed PEAcⅠ mRNA preferentially accumulated in rapidly developing tissues: it peaked in seven days' stem; remained at a rather high level in leaves within a month but decreased significantly later; varied a little in tendrils and reached a median and a very low level respectively in juvenile siliques and in pollen. PEAcⅡ displayed somewhat similar expression pattern to PEAcⅠ. The observed differences in sequences and transcript accumulation patterns suggest that the individual pea actin genes may differ in their transcriptional regulation and cellular function. Phylogenetic tree of actins showed that pea actin isoforms are as diverged from each other as they are from other plant actins, and pea actins might have originated from a common ancestor before the divergence of the dicot and monocot plants.
文摘利用噬菌体衣壳蛋白MS2和带有序列特异性茎环结构(含有MS2蛋白结合位点)的RNA之间的高度亲和力,对外源性人血管紧张素1型受体(angiotensin II receptor type 1,AGTR1)mRNA 3′端非翻译区(3′untranslated region,3′UTR)片段进行红色荧光标记,进而在活细胞(HeLa)内研究该mRNA片段的应激生物学行为。通过在pSG5空载体质粒上先后插入两个双链DNA目的片段AGTR1-3′UTR和24×MS2,构建重组质粒pSG5/AGTR1-3′UTR/24×MS2,并将该质粒与重组质粒pERFP/MS2和pEGFP/C1-G3BP共转染入Hela细胞。荧光显微成像结果显示,AGTR1-3′UTR-24×MS2 mRNA片段能够携带具有入核信号的MS2-RFP融合蛋白离开胞核进入胞浆,而且在亚砷酸盐刺激下,红色荧光标记的AGTR1-3′UTR-24×MS2 mRNA片段可在胞浆中形成与应激蛋白G3BP-GFP共定位的颗粒。该结果表明,针对AGTR1-3′UTR片段的MS2-RFP荧光标记系统构建成功,该荧光标记系统能有效避免假阳性的荧光信号。在细胞受到氧化应激时,AGTR1-3′UTR会被招募至胞浆中的应激颗粒结构中,启示了AGTR1-3′UTR区域对于调控AGTR1 mRNA在细胞内的应激定位具有重要作用。
