摘要
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。
By hiTAIL-PCR（3＇-terminal high-efficiency thermal asymmetric interlaced PCR） and LD-PCR（long distance PCR）, the complete sequence and flanking sequence of T-DNA in transgenie maize D-3 were obtained. A specific PCR detection method for transformant was also established. The results revealed that the total length of T-DNA was 4318 bp, which consisted of a G2-EPSPS gene expression cassette. The primers had been designed based on the 3＇ and 5＇ terminal flanking sequence, followed by specific PCR to detect the sensitivity as well as the detection limit. The results showed that 3＇-qualitative PCR detection method was highly specific and sensitive, with a sensitivity of 0.05% per 100ng DNA template.This research provides the methods for the detection of exogenous gene in transgenic maize and the risk assessment on bio-safety.
出处
《作物杂志》
CAS
北大核心
2016年第1期69-75,共7页
Crops
基金
转基因重大专项(2014ZX0800301B)
国家自然科学基金(31370126)
国家"973"计划(2013CB733903)
国家"863"计划(2012AA063503)
