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通过一次3’端高效热不对称交互PCR(hiTAIL-PCR)和一次长链PCR扩增方法获得了转抗草甘膦基因(G2-EPSPS)玉米品系D-3的外源DNA插入片段的全DNA序列(T-DNA)及两端侧翼序列,并建立了转化体特异性PCR检测方法。结果显示:T-DNA插入片段全长4 318bp,由一个G2-EPSPS基因的表达盒构成。根据T-DNA 5’、3’端侧翼序列设计引物,建立了转化体特异性检测方法,并分析了该方法的灵敏度以及检出限,研究结果表明,3’端定性PCR检测方法特异性强,灵敏度高,检出极限为每100ng模板量的0.05%。本研究结果对转抗草甘膦外源基因检测和生物安全评价及监管具有重要意义。
The complete exogenous DNA fragment of D-3 transformed by glyphosate-resistant gene (G2-EPSPS) maize line was obtained by a 3 ’efficient hiTAIL-PCR and a long PCR amplification method DNA sequence (T-DNA) and flanking flanking sequences were established. A transformant-specific PCR assay was established. The results showed that the T-DNA insert was 4 318 bp in length and consisted of a G2-EPSPS gene expression cassette. Based on the 5 ’and 3’ flanking sequences of T-DNA, primers were designed to detect the transformant specificities and the sensitivity and detection limit of the transformants were analyzed. The results showed that the 3 ’ , High sensitivity, detection limit of 0.05% per 100ng template amount. The results of this study are of great significance for the detection of glyphosate-resistant exogenous genes and biosafety evaluation and regulation.