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以转基因马铃薯株系为材料,采用Real-time PCR方法,以SYBR Green I为荧光染料,以马铃薯块茎贮藏蛋白基因(Patatin)作为内参基因,以植物表达载体上的潮霉素抗性基因(HPT)为筛选标记基因,建立目的基因CT值与起始模版的相关性标准曲线,通过实时荧光定量PCR分别获得每个样品中内参基因和外源基因的CT值,根据Pfaffl法计算获得了T-DNA在转基因马铃薯中的拷贝数,且与Southern blot方法进行了比较,并结合田间农艺性状,分析了外源基因拷贝数马铃薯株高及薯块产量的影响。结果表明,内参基因和外源基因标准曲线的相关系数分别为R2=0.996、R2=0.995和R2=0.990。在所测的23株转基因株系中,12株为单拷贝插入,6株为双拷贝,5株为多拷贝。Real-time PCR比Southern blot方法结果更准确、操作更简便快捷、成本更低。且插入拷贝数的多少与马铃薯田间农艺性状变化有一定的关系,发现2拷贝以上的T-DNA插入较容易引起部分性状的改变。
The transgenic potato lines were used as materials. Real-time PCR method, SYBR Green I as fluorescent dye, and potato tubat storage protein gene (Patatin) as internal reference genes were used to express the hygromycin resistance gene (HPT ) Was used to screen the marker gene and establish the standard curve of the correlation between the CT value of the target gene and the initial template. The CT values of the reference genes and exogenous genes in each sample were obtained by real-time fluorescence quantitative PCR, respectively, and the T- Copy number of DNA in transgenic potato and compared with the Southern blot method, combined with field agronomic traits, analysis of the copy number of exogenous gene copy number of potato and potato yield. The results showed that the correlation coefficients of the reference and reference genes were R2 = 0.996, R2 = 0.995 and R2 = 0.990, respectively. Of the 23 transgenic lines tested, 12 were single copy insertions, 6 were double copies and 5 were multiple copies. Real-time PCR is more accurate than Southern blot results, making it easier and cheaper to operate. There was a certain relationship between the number of inserted copies and the changes of agronomic traits in the field of potato. It was found that the insertion of more than two copies of T-DNA could easily lead to some changes of traits.