利用多重荧光定量PCR检测转基因水稻外源基因拷贝数

发布时间：2018-06-21 03:24

本文选题：转基因水稻 + 拷贝数　；参考：《农业生物技术学报》2017年12期

【摘要】：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。
[Abstract]:The number of copies of foreign genes in transgenic plants affects genetic stability and gene expression level, so it is necessary to identify and screen single-copy homozygous plants for functional analysis of progenies. In order to quickly and accurately identify the copy number of transgenic rice materials containing hygromycin phosphotransferase IIHPT, a hygromycin phosphotransferase gene, In this study, a stable transcription shear factor U2afa Oryza sativa splicing factor U2aftOs SFu2a was used as the internal reference gene to construct the copy number identification system of multiplex PCRX multiplex Q RT-PCR based on Taqman. By comparing the amplification efficiency of different primer combinations, hpt II-1 and Os SFu2a-2 were determined as the best primer combinations. The system was used to identify the copy number of 10 transgenic clones. The results showed that 7 clones were single copy plants and 3 clones were double copy plants. Then, the reliability of the method was verified by Southern blot and the statistics of segregation ratio of transgenic progeny, and compared with the traditional single fluorescence quantitative PCRX Q RT-PCR. The results showed that the detection accuracy and stability were greatly improved by multiplex fluorescence quantitative PCR. Therefore, this method can help researchers quickly, accurately, high-throughput identification of foreign gene copy number and homozygous plants.
【作者单位】：福建省农业科学院水稻研究所;农业部华南杂交水稻种质创新与分子育种重点实验室/福州(国家)水稻改良分中心/福建省作物分子育种工程实验室/福建省水稻分子育种重点实验室/福建省物种质创新与分子育种省部共建国家重点实验室培育基地/杂交水稻国家重点实验室华南研究基地;水稻国家工程实验室;
【基金】：国家转基因农作物新品种培育重大专项(No.2016ZX08001-006和No.2016ZX08001-004) 福建省财政专项-福建省农业科学院创新团队PI项目(No.2016PI-15)
【分类号】：S511

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