转基因大豆、玉米和水稻外源基因检测通用标准分子的构建

Construction of a standard reference plasmid for detecting exogenous genes in transgenic soybean,maize and rice

将转基因大豆、玉米和水稻的主要外源Cry1A(B)基因、BAR基因、CP4-EPSPS基因、PAT基因和内参RBCL基因目标片段分别克隆到克隆载体pMD18-T中,构建获得的质粒可作为定性检测3种转基因粮食作物的上述外源基因的通用标准分子质粒pMD18-T-PAT-CP4-EPSPS-Cry1 A(B)-BAR-RBCL,长约4.7 kb。经过双酶切、测序及PCR扩增,获得与预期片断大小及序列一致的目的基因片段,证明所构建的标准分子质粒是正确的,可以用来作为不同品种转基因粮食作物定性检测CP4-EPSPS基因、Cry1 A(B)基因、BAR基因和PAT基因的通用阳性标准分子。

We cloned Cry 2A （B） , BAR, CP4-EPSPS, PAT and RBCL genes from transgenic soybean, maize and rice into a PMD18-T vector, so that the rseulting novel plasmid, pMD18-T-PAT-CP4-EPSPS-Cry 1A （B）-BAR-RBCL with a full length of 4. 7 kilobase, can be used as the standard reference molecule in the qualitative detection of exogenous genes. Identification by double restriction endonuclease digestion, sequencing and by PCR showed that the recombinant plasmid was in accordance with the expected length and sequence of target genes. Thus, the plasmid constructed in this study is reliable and may be used as a versatile standard reference molecule in the qualitative detection of Cry 2A （B）, BAR, CP4-EPSPS and PAT genes in different transgenic food crops.