更新时间：09-04 （Angela）提供原创文章
摘要：通过分子生物学技术，测定猪背长肌中2个目的基因即维生素E受体基因PXR和共轭亚油酸受体基因PPARγ的mRNA表达水平，分析其相对表达量与猪肉品质的相关性。 方法 根据GenBank中公布的已知PXR、PPARγ基因序列，用Primer Premier 5.0、BLAST等软件设计PCR引物，并与参考的引物进行比对，选择最优引物对。引物经合成后用RT-PCR进行扩增和引物验证。 结果 在所设计的引物中PPARγ引物的扩增效果较好，PXR没有扩增出条带，故最终PXR选用参考引物。 结论 通过探讨和优化PCR体系中退火温度、MgCl2 浓度和循环数等参数，建立PXR、PPARγ目的基因的mRNA表达的半定量RT-PCR体系：PXR最佳扩增条件为退火温度58℃、镁离子浓度2.5mmol、循环数34；PPARγ最佳扩增条件是退火温度59℃、镁离子浓度2.5mmol、循环数31。
关键词：猪背最长肌，PXR，PPARγ， RT-PCR
Abstract:Determine the gene expression of PXR and PPARγ in porcine m.longissimus dorsi by Molecular Biology and analysis the relationship between the expression and porcine tenderness. Methods Based on the known PXR and PPARγ gene sequence reported in GenBank, the primers of which were designed by software such as Primer Premier 5.0, BLAST, etc. Then choice the better one compared with referenced primers. After that, the primers were used to amplified by RT-PCR and could be tested by the experiment. Results Amplified by RT-PCR, PPARγ’ result was better than PXR’s. So the primer of PPARγ was identified to be designed successfully, and we choice the referenced primer of PXR as what we want. Conclusions Set up the better appropriate conditions of PXR and PPARγ in the semi-quantitative RT- PCR system, such as TM,[Mg2+],cycles and so on. The appropriate conditions in the semi-quantitative RT- PCR system of PXR is 58℃,2.5mmol and 34 cycles; and the appropriate conditions of PPARγ is 59℃,2.5mmol and 31cycles.
Key words： PXR, PPARγ, RT-PCR, longissimus dorsi
本实验通过Trizol法提取猪背最长肌中总RNA，并对提取出来的RNA检测后进行逆转录，然后将2个目的基因PXR和PPARγ进行PCR扩增，并对其扩增的三个主要影响因素（即退火温度、[Mg2+]和循环数）进行单因素试验，在单因素试验的基础上进一步进行正交试验，从而得到优化的扩增条件。