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转基因植物中外源基因的拷贝数影响遗传稳定性和基因表达水平,因此鉴定和筛选单拷贝纯合的植株对于子代材料的功能分析是十分必须的。为了快速、准确的对含有潮霉素抗性基因-潮霉素磷酸转移酶II(hygromycin phosphotransferase II,hpt II)的转基因水稻材料进行拷贝数鉴定,本研究选用了稳定的组成型基因水稻转录剪切因子U2af(Oryza sativa splicing factor U2af,Os SFu2a)作为内参基因,构建基于Taqman的多重荧光定量PCR(Multiplex q RT-PCR)的拷贝数鉴定体系。通过不同引物组合进行扩增效率比较,确定了hpt II-1和Os SFu2a-2为最佳引物组合。利用该体系对10个转基因克隆进行了拷贝数鉴定,结果其中7个克隆为单拷贝植株,3个克隆为双拷贝植株。随后,通过Southern blot和转基因子代分离比统计,验证了此方法的可靠性;并通过与传统的单重荧光定量PCR(Singlex q RT-PCR)法进行比较,结果表明多重荧光定量PCR法大大提高了检测的准确性和稳定性。因此,该方法能够帮助科研人员快速,准确,高通量的鉴定水稻外源基因拷贝数与纯合体植株。
The copy number of the foreign gene in the transgenic plant affects the genetic stability and the gene expression level. Therefore, it is very necessary to identify and screen the single copy homozygous plant for the functional analysis of the progeny material. In order to identify the copy number of transgenic rice with hygromycin resistance gene hygromycin phosphotransferase II (hpt II) rapidly and accurately, we selected a stable constitutive gene of rice transcriptional scissors To construct a copy number identification system based on Taqman multiplex multiplex PCR (RT-PCR) with U2af (Osryza sativa splicing factor U2af, Os SFu2a) as an internal control gene. Comparison of amplification efficiency by different primer combinations confirmed that hpt II-1 and Os SFu2a-2 were the best primer combinations. Using this system, the copy number of 10 transgenic clones were identified. Among them, 7 were single-copy and 3 were double-copy. Subsequently, the reliability of this method was verified by Southern blot and transgenic progeny separation ratio statistics. Compared with the traditional Singlex q RT-PCR, the results showed that multiplex PCR method greatly Improve the accuracy and stability of testing. Therefore, this method can help researchers to identify rice homozygous copy number and homozygous plants rapidly, accurately and with high throughput.