马铃薯T-DNA插入拷贝数的检测及对农艺性状的影响研究

Transgene copy number detection and effects on traits of potato(Solanum tuberosum)

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。

Real-time PCR was developed to determine the copy number of exogenous HPTgene in transgenic potato by using SYBR Green I as fluorescent dye.A conserved potato housekeeping gene Patatin was used as an internal control,the standard curves of the cycle threshold（CT）relative to the log of each initial template copy of Patatin and HPT were obtained.The copy number of T-DNA of transgenic potato was calculated according to the method of Pfaffl.And the influence of exogenous gene copy number on plant height and tuber yield of potato was analyzed.The results showed：The coefficients of Patatin and HPT were 0.996,0.995 and 0.990,respectively.Among the twenty three putative transgenic lines,twelve had one copy,six had two copies,and the remaining had multi copies.The method of estimating copy number of exogenous gene by real-time fluorescence quantitative PCR was characterized as convenient,speed and low cost when compared with Southern blot.And there was a correlation between copy number and agronomic traits of potato.