CAJ | 학술논문

　　为了建立洋虫β-actin基因实时荧光定量RT-PCR体系，本实验采用MJ Research OpticonTM 2型实时荧光定量PCR仪，利用SYBR Green I染料法，根据GenBank上其他昆虫β-actin基因的保守序列设计引物，对PCR退火温度、引物浓度、模板浓度等各反应因子进行优化，结合扩增曲线和熔解曲线进行分析。结果显示，在20μL体系下，当2×SYBRR Premix Ex TaqTM为10μL时，引物和cDNA模板的最佳浓度分别为1μmol/L和50 ng/μL。最佳PCR反应程序为：94℃预变性30 s，44个循环包括94℃变性10 s，45℃退火30 s，72℃延伸40 s，最后加做熔解曲线82℃1 s。结果表明，在洋虫不同发育时期β-actin基因表达水平基本稳定，因此β-actin基因可以作为洋虫实时荧光定量RT-PCR的内参基因。本研究成功建立了2-ΔΔCT相对定量法的洋虫β-actin基因实时荧光定量RT-PCR体系，并分析了优化PCR反应体系的重要性，建立的洋虫β-actin基因荧光定量RT-PCR方法简便、特异性强，该体系的建立可用于洋虫蜕皮相关基因表达差异的深入研究。
　　위료건립양충β-actin기인실시형광정량RT-PCR체계，본실험채용MJ Research OpticonTM 2형실시형광정량PCR의，이용SYBR Green I염료법，근거GenBank상기타곤충β-actin기인적보수서렬설계인물，대PCR퇴화온도、인물농도、모판농도등각반응인자진행우화，결합확증곡선화용해곡선진행분석。결과현시，재20μL체계하，당2×SYBRR Premix Ex TaqTM위10μL시，인물화cDNA모판적최가농도분별위1μmol/L화50 ng/μL。최가PCR반응정서위：94℃예변성30 s，44개순배포괄94℃변성10 s，45℃퇴화30 s，72℃연신40 s，최후가주용해곡선82℃1 s。결과표명，재양충불동발육시기β-actin기인표체수평기본은정，인차β-actin기인가이작위양충실시형광정량RT-PCR적내삼기인。본연구성공건립료2-ΔΔCT상대정량법적양충β-actin기인실시형광정량RT-PCR체계，병분석료우화PCR반응체계적중요성，건립적양충β-actin기인형광정량RT-PCR방법간편、특이성강，해체계적건립가용우양충세피상관기인표체차이적심입연구。
In this paper, we constructed a real-time fluorescence quantitative RT-PCR system for theβ-actin gene of Palembus dermestoides with MJ Research OpticonTM 2 instrument, using SYBR Green I method. The primers of β-actin were designed by the conserved sequence of other insects available in GenBank. PCR annealing temperature, primers concentration, template concentration and other reaction factors was optimized, and the amplification curve and melt curve was analyzed. The results showed that the optimum reaction conditions were 1μmol/L of each primer, 50 ng/μL of cDNA templet, and 10μL of 2×SYBRR Premix Ex TaqTM in a final volume of 20 μL. The optimum PCR procedure conditions were initial denaturation 94℃ for 30 s followed by 44 cycles of 94℃for 10 s, 57℃for 30 s and 72℃for 40 s. A melt curve was acquired at 82℃for 1 s finally. It found thatβ-actin could be used as reference gene of FQ RT-PCR of P. dermestoides because of its stable expression during different developmental stages. The 2-ΔΔCT relative quantitative RT-PCR system was established for the β-actin of P. dermestoides and the importance of the optimization of PCR reaction system was analyzed. The FQ RT-PCR method was simple and had a highly specificity and could be further applied to the usage of the β-actin as a reference gene in quantitative analysis of ecdysis relevent gene expression of P. dermestoides.