构建了含有转化体特异性片段（BT11-93bp）和玉米的内标准基因片段（zSSIIb-151bp）的质粒分子pUC57-BT11,经酶切和测序验证后,对其特异性进行检查。采用测序和荧光定量PCR法对该质粒分子进行定值,结果为1.01±0.01(k=2)。通过质粒分子和基因组DNA产生的内源和外源基因比较,发现两者标准曲线斜率无显著差异,线性相关系数均≥0.99。利用pUC57-BT11质粒分子对已知含量的转基因基体标准物质进行转基因含量测定,结果发现采用pUC57-BT11质粒分子对标准物质的测定结果与其标准值一致,表明pUC57-BT11质粒分子可作为转基因玉米BT11转化体特异性的定性和定量检测用标准物质。
Abstract
To construct a plasmid pUC57-BT11 including event specific product and endogenous gene zSSIIb product.After restriction enzyme digestion and sequencing, the specific of the plasmid pUC57-BT11 is detected.The property value of pBT11 is 1.01±0.01(k=2) determined by sequencing and real time PCR.By comparison standard curve generated by plasmid with genomic DNA, no significant difference between the slopes of the standard curve is found.All correlated coefficient R2 are ≥0.99.Certified GM matrix materials are detected by quantitative PCR when pBT11 plasmid DNA is used as quantification standards.The results determined by quantitative PCR with pBT11 are identical with the certified values.This indicated that pBT11 plasmid can be used as a standard for GM maize BT11 quantitative and qualitatively detection.
关键词
计量学 /
转基因玉米 /
BT11 /
质粒标准分子 /
标准物质
Key words
Metrology /
Genetic modified maize /
BT11 /
Plasmid /
Standard material
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参考文献
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［2］GB/T 19495.4—2004.转基因产品检测—核酸定性PCR检测方法［S］.
［3］GB/T 19495.5—2004.转基因产品检测—核酸定量PCR检测方法［S］.
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基金
国家转基因生物新品种培育科技重大专项（2008ZX08012-003）;科技支撑项目2008BAK41B01）
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