肉质相关基因RXR、VDR的扩增_食品论文.rar

摘要：利用半定量逆转录-聚合酶链式反应（RT-PCR）技术，检测猪背最长肌中维生素A受体基因RXR和维生素D受体基因VDR的mRNA表达水平，分析其相对表达量与肉质的关系。方法 通过查找NCBI等数据库中公布的已知RXR和VDR基因序列，用Primer Premier 5.0等软件设计PCR引物，经PCR验证确定最终引物对。引物经合成后进行RT-PCR，对其扩增条件退火温度、Mg2+浓度循环数进行筛选。结果 参考的2对引物各项指标优于设计的引物，故最终选用参考引物。通过探讨和优化PCR体系中退火温度、镁离子浓度和循环数，建立了RXR、VDR受体基因mRNA表达的半定量RT-PCR体系：RXR适宜扩增条件为：59℃，1.5mmol，38循环。VDR最佳条件是59℃，2.0mmol，35循环。

关键词：，RXR，VDR，引物设计，嫩度，猪背最长肌，RT-PCR

Abstract:Determine the gene expression of RXR and VDR in meat longissimus dorsi by RT-PCR method. And analysis the relationship between the expression and meat tenderness. Methods Based on the known RXR and VDR gene sequence reported in NCBI, designed the primer with software such as Primer Premier 5.0 and so on. Then choice the better one compared with referenced primers. After made by biology company, the primers were used to amplified by RT-PCR , and then to optimize MgCl2 concentration, anneal temperature and the number of loop. Results We selected the referenced primers finally as they were better than the ones we designed. By searching the appropriate condition（T,[Mg2+],cycle），set up the semi-quantitative RT-PCR system detecting the mRNA gene expression of RXR and VDR. The appropriate annealing temperature,[Mg2+] and cycle times combination of RXR is 59℃, 1.5mmol,38 cycles. VDR is 59℃, 2.0mmol, 38 cycles.

Key words：RXR, VDR, primer design, meat tenderness, longissimus dorsi RT-PCR

本试验用TRIzol法提取杂交山猪背最长肌中总RNA，进行逆转录，对目的RXR和VDR目的基因进行PCR扩增，并对其扩增条件退火温度、Mg2+浓度和循环数进行单因素，在单因素试验基础上进行正交，得到优化的扩增条件发现不同退火温度、Mg2+浓度和循环数在一定程度上对PCR扩增的效果都有影响。