摘要
为提高烟叶转基因成分检测效率,建立了烤后烟叶中3种外源基因成分的多重PCR检测技术体系,通过优化该反应体系中的Mg2+、d NTP、r Taq酶、引物浓度及退火温度等参数,实验最适条件为将4对相等摩尔浓度引物预先按1∶1∶1∶1(V/V)混合,在Mg2+为1.5 mmol/L、d NTP为125μmol/L、r Taq酶1 U、混合引物1μmol/L,DNA 100 ng,退火温度65℃,循环数35个的反应条件下,在一次PCR反应中便可同时检测烟草内源基因NR,外源基因35S启动子、NOS终止子、NPT II筛选标记基因。优化后的反应体系能够有效地检测出转基因烤后烟叶百分比含量为0.9%(V/V)的转基因成分。
In order to improve the efficiency of detecting genetically modified ingredient ( GMI ) , we established a multiplex PCR detecting 3 exogenous gene ingredients in flue-cured tobacco. By optimizing the concentrations of Mg2+, dNTP, rTaq, and primer as well as annealing temperature in the multiplex PCR system, the result showed that the optimal conditions for the experiment were : 4 pairs of primers pre-mixed in ratio of 1 : 1 : 1 : 1 ( V/V ) , Mg2+ 1.5 mmol/L, dNTP 125μmol/L, rTaq polymerase 1 U, mixed primers 1 μmol/L, DNA 100 ng annealing temperature 65℃, and 35 reaction cycles ; under the above conditions, the system detected 1 tobacco endogenous gene NR, exogenous gene 35S promoter, NOS terminator and selectable marker NPT II gene by 1 PCR reaction. In this study, the optimized multiplex PCR could efficiently detect the GMI of 0.9% ( V/V ) in transgenic flue-cured tobacco.
出处
《生物技术通报》
CAS
CSCD
北大核心
2015年第7期64-68,共5页
Biotechnology Bulletin
基金
贵州省科技支撑计划农业攻关[黔科合NY(2013)3020号]