对南荻MINAC13基因进行了克隆、亚细胞定位和转录激活试验,并用实时荧光定量PCR方法,检测了在盐胁迫、干旱胁迫、低温和脱落酸、茉莉酸甲酯、水杨酸处理下的MlNAC13基因在南荻根部的表达模式。结果:1)获得了南荻MINAC13的全基因序列;2)用N端融合YFP进行亚细胞定位试验的结果证明,MlNAC13位于细胞核,MlNAC13可能在细胞核内行使功能;3)MlNAC13的C端及全长具有转录激活活性,N端没有转录激活活性,说明它是转录因子,且转录激活域位于C端;4)除水杨酸外,脱落酸、茉莉酸甲酯均能诱导MlNAC13基因的表达,说明MlNAC13基因可能参与南荻的多种逆境胁迫响应。

MlNA C13 gene was cloned from Miscanthus lutarioriparius (M.lutarioriparius),and subcellular localization and transactivation assay were conducted on this gene.Then real-time quantitative PCR (RT-qPCR) was used to analyze the expression profile of MlNAC13 in root under salt,drought,cold,abscisic acid (ABA),Methyl jasmonate (MeJA) and Salicylic acid (SA) treatments.The results showed that 1) complete MlNAC13 gene sequence of M.lutaroriparius was obtained; 2) subcellular localization analysis using N-terminus fused with YFP showed that MlNAC13 was located in nucleus,suggesting that it might function in nucleus; 3) transactivation assay indicated that the full-length and C-terminus of MlNAC13 had transactivation activity,indicating that M1NAC13 is a transcription factor and the transactivation activity region located in C-terminus; 4) the transcript of MlNAC13 was up-regulated under all surveyed treatments except SA,indicating that it might participate in various stress responses ofM.luta

目的利用TALE-TFs,在小鼠成纤维细胞中激活β-酪蛋白基因启动子,为检测β-酪蛋白基因启动子—目的基因表达框的表达结果,提供一种检测途径。方法将构建的TALE-TFs和β-酪蛋白基因启动子—Red报告基因质粒电转染进入小鼠成纤维细胞,通过荧光显微镜直接观察报告基因表达情况。结果与结论利用TALE人工转录因子,在小鼠成纤维细胞中能够激活β-酪蛋白基因启动子表达框,为替代乳腺上皮细胞表达验证系统提供了新的途径。

Objective TALE-TFs were adopted to provide a new way in detection of the expression result ofβ-ca-sein gene promoter-interesting gene expression cassettes in mouse fibroblasts.Methods TALE-TFs of eukaryotic expres-sion plasmid and expression cassette withβ-casein gene promoter and red fluorescent protein reporter gene were co-nucleo-fected into mouse fibroblasts by Amaxa nucleofector.Results and Conclusion β-casein gene promoter was activated by artificial TALE-TFs in the mouse fibroblasts.The way is a new expression verification system instead of mammary epithelial cells with fibroblasts.

探讨降脂药物非诺贝特激活法尼醇X受体FXR的机制。方法 :分离小鼠肝脏原代细胞,予非诺贝特处理,通过实时定量PCR检测FXR下游基因的表达情况,并通过荧光素酶双报告基因实验,分析非诺贝特对FXR下游基因调控的机制。结果:①非诺贝特处理可诱导FXR下游靶基因SHP和BSEP的表达,进而降低三酰甘油合成关键转录因子SREBP1c的表达。②非诺贝特能促进FXR上调SHP和BSEP启动子活性,而FXR配体结合区域及转录激活域缺失型则丧失该功能。结论:非诺贝特可能是潜在的FXR激动型配体,可通过激活FXR信号通路,降低三酰甘油合成基因的表达,从而抑制肝脏三酰甘油的沉积。

Objective: To investigate the mechanism of fenofibrate in regulating farnesoid X receptor (FXR) transcriptional activity. Methods:Mouse primary hepatocytes (MPH) were cultured and treated with fenofibrate or vehicle as control. Cells were harvested for RNA isolation and real-time PCR was performed to analyze expression levels of FXR down-stream gene, including SHP, BSEP and SREBP1c. Besides, transcriptional activity of FXR was determined by reporter luciferase assays. Results: Fenofibrate up-regulated mRNA levels of SHP and BSEP, and down-regulated expression of SREBP1c. Fenofibrate could transactivate promoter activity of SHP and BSEP in the presence of full-length FXR, but not the one with ligand-binding-domain or transactivation domain-2 deletion. Conclusions: Fenofibrate might be a potential agonist of FXR,and it could inhibit hepatic triglyceride accumulation by activating FXR signalling and down-regulating lipogenic genes.

目的：研究乳腺癌细胞中TANK结合激酶1（TBK1）的功能及分子机制。方法：利用含有雌激素应答元件（ERE）的萤光素酶报告基因检测TBK1对雌激素受体α（ERα）转录活性的影响；将TBK1参与激活先天免疫途径的关键位点突变，使其丧失激活先天免疫的功能，再用同样方法检测TBK1突变体对ERα转录活性的影响；采用RT-PCR检测TBK1通过磷酸化修饰对ERα下游基因表达水平的影响。结果：TBK1增强ERα的转录活性，从而增强其下游基因的表达。结论：TBK1能以不依赖于其激活先天免疫途径功能的方式增强ERα的转录活性。

Objective: To explore the function of human TANK-binding kinase 1(TBK1) during the breast can?cer development. Methods: We used a luciferase reporter assay system to detect the transcriptional activity of es?trogen receptor α(ERα), the same system to test the function of TBK1 native-immunodeficiency mutant, and then RT-PCR method to test the effect of TBK1 on ERα target genes. Results: TBK1 increased ERα transcriptional ac?tivity and promoted expression of ERα-downstream genes. Conclusion: TBK1 increased ERα transcriptional activi?ty independent of its role in native immune.

【目的】从目前已知的参与拟南芥Arabidopsis thaliana次生壁加厚生长的转录因子着手,分析这些次生壁相关的转录因子是否能够调控木糖合成关键酶基因FRA8、IRX9、IRX10、IRX14、F8H、IRX9-L、IRX10-L和IRX14-L的表达,并且观察KNAT7基因显性抑制植株的表型.【方法】通过Gateway技术构建效应器和报告器,进行瞬时转录激活试验,同样构建pCAMBIA1304-p35S∷KNAT7-SRDX重组质粒,用农杆菌Agrobacterium tumefaciens花序浸染法将此质粒转化到野生型拟南芥植株中.【结果和结论】瞬时转录激活试验表明,转录因子KNAT7、MYB46、ERF72、SND1、NST2能够激活多个拟南芥木聚糖合成关键酶基因的表达,其中KNAT7能促进基因FRA8、IRX9和IRX14-L的表达.KNAT7基因显性抑制能显著影响拟南芥的生长.试验结果表明KNAT7基因可能在木聚糖的合成中起着重要的调控作用.

Objective] To analyze whether some transcription factors in Arabidopsis thaliana, known for the secondary cell wall thicken , could regulate the expression of the key genes of xylosyltransferase , such as FRA8, IRX9, IRX10, IRX14, F8H, IRX9-L, IRX10-L and IRX14-L, and observe the phenotype of KNAT7 dominant repression plant .[Method] Effectors and reporters were constructed by Gateway Tech-nology and the transient transcriptional activation assay was conducted .Construct pCAMBIA1304-p35S∷KNAT7-SRDX recombinant plasmid by Gateway Technology and transform this plasmid into wild A.thali-ana via Agrobacterium tumefaciens-floral dip method .[Result and conclusion] The transient transcription-al activation assay revealed that transcription factors KNAT 7, MYB46, ERF72, SND1, NST2 could acti-vate the expression of a number of the key genes of xylosyltransferase .KNAT7 could activate the expres-sion of FRA8, IRX9 and IRX14-L.Furthermore, dominant repression of KNAT7 significantly affected the