论文部分内容阅读
目的 对GBP130的 5′近端侧翼序列进行分析 ,发现可能的强度和期特异性调控元件。 方法 构建含有不同长度GBP130启动子的质粒载体。用pGBPCATΔ 2、pGBPΔ2 /4 0 0和 pGBPΔ2 /80 0分别转染疟原虫 ,在转染后 48h收集虫体制备细胞抽提物 ,检测报告分子CAT活性 ,分析近端侧翼序列不同区域的强度调控功能。另将 pGBPΔ2 /4 0 0和pGBPΔ2 /80 0分别同时转染疟原虫 ,分别于转染后 5h、15h和 46h收集虫体 ,制备细胞抽提物 ,检测CAT活性 ,分析近端侧翼序列不同区域的期特异性调控功能。 结果 强度分析中 ,当从转录起始点下游删除较小的片段 ( pGBPΔ2 /80 0 ) ,CAT表达水平与pGBPCATΔ 2中CAT水平无显著性差异 (P >0 .0 5 ) ,当删除近端较大片段 ( pGBPΔ2 /4 0 0 )时 ,CAT表达水平明显下降 (P <0 .0 5 )。同时 pGBPΔ2 /4 0 0的转录活性与对照组也有显著性差异 (P <0 .0 5 ) ;期特异性分析 ,pGBPΔ2 /4 0 0与pGBPΔ2 /80 0相比 ,在 5h时前者转录活性高于后者 ,但在 15h和 46h时 ,后者转录活性高于前者 ,提示 2种质粒在疟原虫不同生活阶段具有期特异性调控。 结论 推测不同GBP130启动子活性强度的差异是因为 5′UTR长度的不同 ,较长的UTR可促进基因的转录表达 ,可能GBP130基因近端侧翼序列中 2个同聚 po
Objective To analyze the 5 ’proximal flanking sequence of GBP130 and to find possible intensity and stage-specific regulatory elements. Methods Plasmid vectors containing different lengths of GBP130 promoter were constructed. The plasmids were transfected with pGBPCATΔ2, pGBPΔ2 / 400 and pGBPΔ2 / 80 0 respectively. The cell extracts were collected 48 h after transfection to detect CAT activity. The intensity of different regions of proximal flanking sequences was analyzed Features. In addition, pGBPΔ2 / 400 and pGBPΔ2 / 80 0 were transfected into Plasmodium at the same time. The parasites were harvested at 5h, 15h and 46h after transfection respectively. Cell extracts were prepared and CAT activities were assayed. The different regions of proximal flanking sequence Period-specific regulatory function. In the results of intensity analysis, when the smaller fragment (pGBPΔ2 / 80 0) was deleted from the downstream of the transcription start point, there was no significant difference in the CAT expression level between pGBPCATΔ2 and CAT (P> 0.05) Large fragment (pGBPΔ2 / 400) CAT expression was significantly decreased (P <0.05). At the same time, the transcriptional activity of pGBPΔ2 / 400 was also significantly different from the control group (P <0.05). The specificity of pGBPΔ2 / 400 was higher than that of pGBPΔ2 / 80 0 at 5h The latter, but at 15h and 46h, the latter transcriptional activity was higher than the former, suggesting that the two plasmids in different stages of life-stage with period-specific regulation. Conclusions The difference in the activity of different GBP130 promoters is due to the different length of 5’UTR. The longer UTR can promote the transcriptional expression of genes. It is probable that two of the proximal flanking sequences of GBP130 gene are homopolymeric po