线粒体转录复合物各因子质粒标准品的构建  

Construction of each plasmid standard of the mitochondrial transcription complex

作　　者：罗德艳[1] 冯俊[1] LUO De -yan;FENG Jun(Department o f Otolaryngology ,Nanchong Central Hospital, The Second Clinical College o f North Sichuan Medical College, Nanchong 637000 , Sichuan, China)

机构地区：[1]川北医学院第二临床医学院.南充中心医院耳鼻喉科,四川南充637000

出　　处：《川北医学院学报》2016年第3期392-395,共4页Journal of North Sichuan Medical College

基　　金：四川省教育厅项目(13ZB0241);南充市科技局项目(13A0054)

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、TFB1M、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c DNA,PCR扩增、纯化目的片段,将纯化产物与p Zero Back/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 innerear, the plasmid standards of mitochondrial transcription factor TFAM (A ) , mitochondrial transcription TFB1M ( B1 ) , mitochondrialtranscription TFB2M ( B 2) and mitochondrial RNA polymerase (POLRMT) of rat were constructed. M ethods : Specific primers andprobes 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 establishrecombined plasmid. The recombinant plasmids picked out from positive clones were identified by PCR and then sequenced. Using thepositive clones, the standards curve of target gene recombinant plasmids were constructed by real-time PCR. R esults:The sequencing resultswere consistent with the objective genes and objective standard curves perfected could be received(i?2 > 0 .9 9 ). Conclusion:Theplasmid standard for each target gene was successfully constructed.

关 键 词：线粒体转录因子A 线粒体转录因子B1 线粒体转录B2 线粒体RNA聚合酶 质粒标准品 实时荧光定量PCR 

分 类 号：R332[医药卫生—人体生理学]