更新时间：09-04 （Angela）提供原创文章
摘要：利用半定量逆转录-聚合酶链式反应技术，测定猪背最长肌中PRKAG3、RYR1 基因的mRNA表达，确定二者扩增的适宜条件。方法 根据GenBank 中公布的已知 PRKAG3、RYR1 基因序列，用Primer Primer 5.0、DNAMAN6.0、BLAST 、Oligo6 等软件设计PCR引物，提取猪背最长肌中mRNA 进行逆转录，用RT-PCR 方法进行扩增条件的优化。结果 确定了目的基因PRKAG3 和RYR1 基因的扩增适宜条件。结论 通过探讨PCR 体系中退火温度、MgCl2 浓度和循环次数等参数，建立了检测猪肉肉色相关基因PRKAG3、RYR1 表达的半定量RT-PCR 体系，摸索出了PCR 反应的最优条件，RYR1 退火温度为56℃、[Mg2+] 为1.0 mmol、循环数为35； PRKAG3 为退火温度57℃、[Mg2+] 为2 mmol、循环数为38。
关键词：RT-PCR，RYR1，PRKAG3，引物设计，背最长肌，肉色
Abstract:Objective Determine the gene expression of PRKAG3、RYR1 in porcine m. longissimus dorsi by RT-PCR method, and analysis the fitting condition of them. Methods Based on the known PRKAG3 and RYR1 gene sequence reported in GenBank, the primers of which were designed by software such as Primer Premier 5.0, BLAST, DNAMAN 6.0, Oligo6, and so on. After made by biology company ,the primers was used to amplified by RT-PCR, which can also test the primers. Amplified by RT-PCR, the result was good. So the primer of PRKAG3 and RYR1 were identified to be designed successfully. Conclusions The semi - quantitative RT - PCR system detecting the mRNA gene expression of PRKAG3 and RYR1 were set up by discussing and optimizing MgCl2 concentration, anneal temperature and the number of loop and finally find the best situation of them. The appropriate annealing temperature﹑[Mg2+] and cycle times combination of PRKAG3 was 57℃、2.0 mmol、38 cycles；through interactive experiment the appropriate temperature，[Mg2+] and cycle of RYR1 was 56℃、1.0mmol、35 cycles.
Key words: RT-PCR, RYR1, PRKAG3, primer design, longissimus dorsi, meat color
本实验提取杂交山猪背最长肌中总RNA经检验后发现符合要求，对其进行逆转录，并对PRKAG3和RYR1目的基因进行PCR条件筛选，筛选的方法主要是先对其扩增条件包括退火温度、[Mg2+]和循环数进行单因素试验，在单因素实验基础上选择最佳的一个条件进行三因素三水平的正交试验，得到最佳的扩增条件组合。主要结论如下：
1 经过查阅文献并实验验证确立了目的基因RYR1、PRKAG3 的引物序列。
2 用半定量RT-PCR技术，对目的基因的扩增条件中的退火温度、[Mg2+] 和循环数进行梯度优化，得到目的基因的适宜扩增条件为：
RYR1退火温度为56℃、[Mg2+] 为1.0 mmol、循环数为35。
PRKAG3为退火温度57℃、[Mg2+] 为2.0 mmol、循环数为38。