摘要
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 GreenⅠ real-time PCR was developed to determine the copy number of exogenous pta gene in transgenic wheat.A conserved wheat housekeeping gene,wx012,was used as an internal control and 5 times diluted non-transgenic wheat genome DNA is used as an initial template copy of wx012.So the y=-0.2667x＋6.98,the standard curves of the cycle threshold （CT ） relative to the log of each initial template copy of wx012 was obtained.In the same way,the standard curves of pta y=-0.2118x＋4.53 is obtained using 5 times diluted plasmid DNA which contains pta gene.By SYBR GreenⅠ real-time PCR the CT values of wx012 and pta gene in each transgenic wheat was got,the transgene copy number is calculated by comparing the initial template copy of pta to wx012 gene in the same transgenic wheat.The conclusion is,among the seven PCR putative transgenic plants：one was non-transgenic plant,one had one copy number,one had two copies,and the other had three,four copies,respectively.
出处
《中国生物工程杂志》
CAS
CSCD
北大核心
2010年第3期90-94,共5页
China Biotechnology
基金
甘肃省科技攻关计划项目(2GS035-A41-001-03)资助项目
