SYBR Green实时定量PCR检测转基因大豆中外源基因拷贝数  

Establishment of SYBR Green-base quantitative real-time PCR assay for determining transgene copy number in transgenic soybean

作　　者：冀志庚[1] 高学军[1] 敖金霞[1] 张明辉[1] 霍楠[1] 

机构地区：[1]东北农业大学生命科学与生物技术研究中心,哈尔滨150030

出　　处：《东北农业大学学报》2011年第10期11-15,共5页Journal of Northeast Agricultural University

基　　金：国家转基因重大专项课题(2008ZX08012-001)

摘　　要：采用SYBRGreen Ι real-time PCR方法检测转基因大豆中外源基因35S边界基因的拷贝数,以大豆凝集素基因(Lectin)作为内参照基因,以转基因大豆基因组DNA为内参照基因标准品,初始浓度为0.43μg·μL-1,进行5倍梯度稀释得到内参照基因CT值与起始模板量的相关性标准曲线:y=-2.9915x+22.3321,R2=0.996;以含35S边界序列的质粒DNA为目的基因为标准品,初始浓度为0.64μg·μL-1,同样进行5倍梯度稀释,建立目的基因CT与起始模板量的相关性标准曲线:y=-3.4021x+17.7219,R2=0.999。通过SYBR Green Ιreal-time PCR获得样本中目的基因和内参基因的CT值,将CT值分别代入标准曲线计算该样本中内参基因和目的基因起始模板量,目的基因与内参基因起始模板量比值即是目的基因在该转基因中的拷贝数。计算结果为4份样品中,2个拷贝的1个,3个拷贝的3个。SYBR Green I real-time PCR was developed to determine copy number of exogenous 35S/ plant gene in transgenic soybean. A conserved soybean housekeeping gene, Lectin was used as an internal contral and 5 times diluted soybean genome DNA was used as an initial template copy of Lectin. So y=-2.9915x＋22.3321, R2=0.996, the standard curves of the cycle threshold （CT） relative to the log of each initial template copy of Lectin was obtained. In the same way, the standard curves of 35S/plant y=-3.4021x＋ 17.7219, R2=0.999, was obtained using 5 times diluted plasmid DNA which contains 35S/plant gene. By SYBR Green I real-time the CT values of Lectin and 35S/plant gene in each transgenic soybean was got, the transgene copy number was calculated by comparing the initial template copy of 35S/plant to Lectin gene in the same transgenic soybean. It concluded that among four PCR putative transgenic plants： one had two copies, and the other had three copies, respectively.

关 键 词：real-time PCR SYBR Green Ι 转基因大豆 拷贝数 LECTIN 35边界序列 

分 类 号：S565.1[农业科学—作物学] Q78[生物学—分子生物学]