线粒体转录复合物各因子质粒标准品的构建

摘要

目的：构建大鼠线粒体转录因子 TFAM（A）、线粒体转录因子 TFB1M （B1）、线粒体转录 TFB2M （B2）及线粒体RNA 聚合酶（POLRMT）的质粒标准品，为检测内耳细胞线粒体 TFAM、TFB1 M、TFB2M、POLRMT 的 mRNA 表达水平做基础。方法：设计特异性引物和探针，提取大鼠内耳组织总 mRNA 逆转录成 cDNA，PCR 扩增、纯化目的片段，将纯化产物与 pZer-oBack ／blunt 载体重组，提取重组质粒，经测序鉴定后，用实时荧光绝对定量 PCR 建立标准曲线。结果：测序结果与各目的序列一致，获得良好的标准曲线（R2＞0．99）。结论：成功构建了各目的基因的质粒标准品。%Objective:To form the basis of detecting the expression of TFAM,TFB1M,TFB2M and POLRMT mRNA of inner ear,the plasmid standards of mitochondrial transcription factor TFAM (A),mitochondrial transcription TFB1 M (B1 ),mitochondrial transcription TFB2M (B2)and mitochondrial RNA polymerase (POLRMT)of rat were constructed.Methods:Specific primers and probes were designed,total mRNA was extracted from rat inner ear tissues and reverse transcribed to cDNA using the specific primers. Purified target fragments from amplified objective gene sequences through PCR were transformed to pZeroBack /blunt vector to establish recombined plasmid.The recombinant plasmids picked out from positive clones were identified by PCR and then sequenced.Using the positive clones,the standards curve of target gene recombinant plasmids were constructed by real-time PCR.Results:The sequencing re-sults were consistent with the objective genes and objective standard curves perfected could be received(R2 >0.99).Conclusion:The plasmid standard for each target gene was successfully constructed.