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在已获得水稻泛素连接酶基因OsHUB2过表达转基因植株的基础上,根据OsHUB2基因序列设计3对引物,分别为OsHUB2-1、OsHUB2-2、OsHUB2-3,通过RT-PCR筛选条带特异单一的引物,real time PCR(qRT-PCR)方法验证最适合检测OsHUB2的反应条件。结果表明OsHUB2-F3R3扩增的PCR产物特异性最强,扩增曲线及熔解曲线等指标都在理想范围内,同时该引物能很好地区分对照及转基因材料,进而筛选出OsHUB2基因上调表达的转基因植株。Realtime体系的建立与优化为今后进一步研究水稻泛素连接酶基因OsHUB2的功能奠定了基础。
Based on the OsHUB2 overexpression transgenic rice plants, three pairs of primers were designed according to the OsHUB2 gene sequence, which were OsHUB2-1, OsHUB2-2 and OsHUB2-3, respectively, and the bands were identified by RT-PCR. The real-time PCR (qRT-PCR) method was used to validate the optimal conditions for the detection of OsHUB2. The results showed that the PCR products amplified by OsHUB2-F3R3 had the strongest specificity and the amplification curves and melting curves were within the ideal range. At the same time, the primers could well distinguish between the control and transgenic materials, and then screened OsHUB2 up-regulated Transgenic plants. The establishment and optimization of Realtime system lays the foundation for further study on the function of OsHUB2, an ubiquitin ligase gene in rice.