转基因大豆中外源基因的二重PCR检测

The detection of exogenous genes with duplex PCR in genetically modified soybean line Roundup Ready

用CTAB法提取转基因大豆(Roundup Ready品系)的总DNA.根据行业标准SN/T 1195-2003,合成用于扩增花椰菜花叶病毒(Cauliflowerm osaic viru,s CaMV)35S启动子的引物S1和S2、根癌农杆菌(Agrobacterium tum efaciens)CP4菌株EPSPS基因的引物Ea、ER和Eb.应用这5条(2组)引物对转基因大豆进行二重PCR检测时,扩增得到2个新片段,分别与片段S1ER、S1Eb大小一致.将片段S1ER、S1Eb进行克隆并测序,序列已在GenBank上登录,接受号分别为AY592954和AY596948.序列分析结果表明,片段S1ER含有CaMV 35S启动子和矮牵牛叶绿体转运肽序列(CTP)的部分序列,片段S1Eb含有CaMV 35S启动子部分序列、CTP和CP4 EPSPS基因的部分序列,原用于扩增CP4 EPSPS基因的引物中,仅引物Eb位于CP4 EPSPS基因中.重新合成扩增CP4 EPSPS基因的引物E1和E2,建立的二重PCR方法不仅适合于转基因RoundupReady大豆,还适合于其它植物中的CaMV 35S启动子和CP4 EPSPS基因的同时检测.

The DNA of genetically modified soybean line Roundup Ready was extracted by CTAB method. The primers were synthesized according to entry-exit inspection and quarantine industry standard SN/T1195-2003. S1 and S2 were used to amplify 35S promoter of cauliflower mosaic virus （CaMV）, while Ea, ER and Eb were used to amplify CP4 EPSPS gene of Agrobacterium tumefaciens strain. As we tried the duplex PCR with the 5 primers （2 groups） to detect synchronously CaMV 35S promotor and CP4 EPSPS gene in the soybean line Roundup Ready, two new fragments were amplified and their sizes were the same as those of fragments S1ER and S1Eb. The fragments S1ER and S1Eb were cloned and sequenced, and the sequences were accepted by GenBank as AY592954 and AY596948, respectively. The sequence analysis showed that fragment S1ER included partial sequence of CaMV 35S promoter and chloroplast transit peptide（CTP） from Fetunia hybrida, while the fragment S1Eb consisted of partial sequence of CaMV 35S promoter, CTP and partial sequence of CP4 EPSPS gene. In fact, only primer Eb was located inside the ORF of CP4 EPSPS gene. The new primers E1 and E2 of CP4 EPSPS gene were synthesized, and the duplex PCR method synchronously detecting CaMV 35S promoter and CP4 EPSPS gene was suitable for genetically modified soybean and other plants.