摘要
GTS40-3-2是抗草甘膦转基因大豆,为建立GTS40-3-2大豆转化体特异性PCR检测方法,本研究以GTS40-3-2标准品为实验材料,根据已公布转基因大豆GTS40-3-2基因与大豆基因组连接序列信息,利用Primer5.0软件设计了5对品系特异性引物,对每对引物进行了退火温度、特异性及扩增效率的PCR检测,结果显示,5对特异性引物均能够从GTS40-3-2中扩增出大小约279bp、238bp、470bp、490bp和257bp的预期产物,可用于特异性检测转基因大豆GTS40-3-2转化事件。以转基因大豆GTS40-3-2含量为5%、2%、1%、0.5%和0.1%的标准品进行PCR灵敏度检测,结果表明5对引物的检测灵敏度均能达到0.1%。通过荧光定量PCR对5对特异性引物的Ct值与溶解曲线比较,最后选择出RRS2引物对为转基因大豆GTS40-3-2品系特异性检测的最适引物。本文结果将为我国未来转基因生物产品成分检测提供科学合理的实验参考。
GTS40-3-2 is a glyphosate-tolerant transgenic soybean. In order to establish a specific PCR validation method for transgenic soybean GTS40-3-2, in this research, we employed GTS40-3-2 standard sample as the experiment material, and designed five specific PCR primers based on the junction sequence published between transgenic soybean gene GTS40-3-2 and soybean genome by using Primer 5.0 software to PCR validation the Tm value, specific and the amplification efficiencies from each primer. The results showed that the products size of five primers was in accordance with expected one which was 279 bp, 238 bp, 470 bp, 490 bp and 257 bp respectively from the standard reference GTS40-3-2 and they can be used for specific validation of transformation event in transgenic soybean GTS40-3-2. Further, we utilized the content of 5%, 2%, 1%, 0.5% and 0.1% transgenic soybean GTS40-3-2 as the sample to carry out PCR sensitivity validation, the results demonstrated the sensitivity of the five primers could reach 0.1%. Finally, we selected out RRS2 primer was the best primer for specific validation of transformation event in transgenic soybean GTS40-3-2 by using fluorescent quantitative PCR in comparison with Ct values and the melting curves among five specific Primers. The results of this paper would provide a scientific and reasonable experiment refference for validating the composition of genetic modified organisms in the future in our country.
出处
《基因组学与应用生物学》
CAS
CSCD
北大核心
2010年第6期1177-1183,共7页
Genomics and Applied Biology
基金
现代农业产业技术体系建设专项资金(nycytx-24)
国家948项目(2006-G37
2010-S19)
国家基金项目(31070330)
国家"863"计划项目(2007AA100701)
福建省科技计划重点项目(2007I0036)
福建省自然科学基金项目(2009J05050)
福建科技项目备案(F2007AA100701)共同资助
