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采用SYBR GreenⅠreal-time PCR方法检测7株转基因小麦中外源半夏凝集素基因的拷贝数。以小麦蜡质基因(wx012)作为内参基因,以未转基因小麦基因组DNA为内参基因标准品进行5倍梯度稀释得到内参基因CT值与起始模板量的相关性标准曲线:y=-0.2667x+6.98;以含半夏凝集素基因(pta)的质粒DNA为目的基的因标准品同样进行5倍梯度稀释,建立目的基因CT值与起始模板量的相关性标准曲线:y=-0.2118x+4.53。通过SYBR GreenⅠreal-time PCR分别获得每一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植株中的拷贝数。计算结果为:单拷贝的有1株,2个拷贝1株,3拷贝和4拷贝的各有2株,其中有1株为假阳性植株。
SYBR GreenⅠreal-time PCR method was used to detect the copy number of the exogenous rhizome lectin gene in seven transgenic wheat lines. The standard curve of correlation coefficient between CT value and initial template was obtained by using wheat waxy gene (wx012) as an internal control gene and non-transgenic wheat genomic DNA as a reference standard for 5-fold dilution. Y = -0.2667x + 6.98; The standard DNA of the pta-containing plasmid DNA was also subjected to 5-fold gradient dilution to establish a standard curve of correlation between the CT value of the target gene and the amount of the initial template: y = -0.2118x +4.53. The CT values of the target gene and the reference gene in each sample were obtained respectively by SYBR GreenⅠreal-time PCR, and the CT values were respectively substituted into the standard curve to calculate the initial template amount of the reference gene and the target gene in the sample. The target gene and the reference gene start template The amount ratio is the copy number of the target gene in the transgenic plant. The results are as follows: 1 copy of single copy, 1 copy of 2 copies, 2 copies of 3 copies and 4 copies, of which 1 is false-positive.