【作者】 袁磊；

【导师】 赵蕾； 路兴波；

【作者基本信息】 山东师范大学 ， 食品科学， 2010， 硕士

【摘要】 MON88017是Monsanto公司通过农杆菌介导转化方法,将含有cry3Bb1和cp4 epsps两个基因表达盒的质粒载体PV-ZMIR39导入玉米Hi-II细胞中得到的转基因玉米品系。对MON88017的分子生物学分析证实,只有单一拷贝的cp4 epsps和cry3Bb1基因表达盒整合到MON 88017的基因组中的单一位点上;各种表达元素完好无损;没有任何细菌质粒骨架序列插入MON88017基因组。本实验采用基因组步移法和巢式PCR方法测定出MON88017外源DNA导入位点旁侧序列,并根据旁侧序列设计转化体特异性(event specific)PCR检测引物,进行定性PCR扩增,确定了引物的最佳反应条件,从而建立起该转基因玉米品系的转化体特异性检测方法。在定性PCR基础上,设计合适的引物和探针,利用定量PCR扩增仪进行荧光实时定量PCR扩增,通过与标准曲线比对,确定产品中转基因成分含量,从而建立起一套完整的抗虫抗除草剂玉米MON88017及其产品转基因成分定量检测体系。详细结果如下:(1)采用基因组步移法和巢式PCR方法获得转基因玉米MON88017外源基因插入位点的左边界旁侧序列504bp。序列同源性分析表明,从5’端开始前168bp为玉米基因组序列,从第169bp至3’末端为插入到玉米中的载体序列,其中从第169bp~479bp为间插序列,从第480bp至3’末端为水稻中启动子P-ract1部分序列。(2)根据转基因玉米MON88017左边界序列,采用Primer premier 5.0软件设计了3对MON 88017转化体特异性引物,对3对特异性引物进行筛选和反应条件优化,确定最适的特异性引物MON88017-1F/R(446bp),其最适退火温度为56℃。通过验证,该引物MON88017-1F/R具有较高的特异性和准确性。用特异性引物MON88017-1F/R对不同浓度的DNA进行检测表明,在MON88017 DNA稀释至0.1%时,仍能扩增出446 bp的特异性目的片段,说明该转基因玉米的最低检测极限(即灵敏度)较高,为0.1%。该定性PCR方法完全可以满足转基因玉米品系的转化体特异性检测的要求。(3)根据转基因玉米MON88017左边界序列,采用Primer Express2.0软件设计了转化体特异性定量引物MON88017-F/R(87bp)和Taqman探针MON88017-P,通过筛选和反应条件优化,确定其最适退火温度为60℃。进行实时荧光定量PCR检测,Taqman探针法定量PCR检测结果显示,0.01%的转基因含量可检测出,灵敏度高于定性PCR检测方法,表明所设计的定量PCR引物、探针特异性好,所建立的实时荧光定量PCR检测体系准确、可靠、灵敏度高。更多还原

【Abstract】 MON88017 was a kind of genetically modified maize discovered by the Monsanto Company through Agrobacterium mediated transformation method, which had transferred the plasmid vector PV-ZMIR39 containing two genes cry3Bb1 and cp4 epsps expression cassette into maize Hi-II cells. Molecular biological analysis on MON88017 confirmed that there was only a single copy of cp4 epsps and cry3Bb1 gene cassette integrated into a single point of the genome of MON 88017; various expression elements were intact; there was no bacterial plasmid backbone sequences inserted into MON88017 genome.In this study the genome walking method and nested PCR method were employed to determine the lead-in sites flanking sequence of MON88017 exogenous DNA, and flanking sequences of transformants under specific (event specific) PCR detection primers are used for qualitative PCR, further to determine the optimum conditions of primers so as to establish the transgenic maize lines transformants PCR method. Based on the Qualitative PCR, appropriate primers and probes were designed, and with the help of quantitative fluorescent real-time quantitative PCR, compared with the standard curve to determine the content of genetically modified products, a comprehensive anti-insect and herbicide-resistant corn MON88017 system and its quantitative detection of genetically modified products were established. Detailed results were as follows:(1) The left border flanking sequence 504bp of transgenic maize MON88017 foreign gene insertion site was obtained through the genome walking method and nested PCR method. Sequence analysis showed that from the 5’ end to 168bp lays the maize genome sequence, and from 169bp to 3’ end was the vector sequence that was inserted into the corn, among which 169bp~479bp were inserted sequences, while from 480bp to 3’ end were promoter sequences P-ract1 for the rice.(2) According to the left border sequences of GM maize MON88017, three pairs of MON 88017 transformants specific primers were designed with Primer premier 5.0 software, and the three pairs of specific primers were screened and optimized with reaction conditions. The optimal specific primers MON88017-1F/R (446bp) were determined, and the optimal annealing temperature was 56℃. Through validation, the primers MON88017-1F/R had a high specificity and accuracy. Tests on different concentrations of DNA with specific primers MON88017-1F/R showed that when MON88017 DNA was diluted to 0.1%, specific fragment of 446bp could still be amplified, indicating that the transgenic maize minimum detection limit (i.e., sensitivity) was high to 0.1%. The qualitative PCR method could meet the specific detection requirements of transgenic maize lines transformants.(3) According to the left border sequences of GM maize MON88017, transformants specific quantitative primer MON88017-F/R (87bp) and Taqman probe MON88017-P were designed with Primer Express 2.0 software, through the screening and optimization of reaction conditions, the optimal annealing temperature was determined as 60℃. Real-time fluorescence quantitative PCR detection was conducted and Taqman probe quantitative PCR results showed that 0.01% of GM content could be detected. Therefore, it’s more sensitive than qualitative PCR detection method. The results indicated that the quantitative PCR primers and probes were quite specific and the established Real-time fluorescence quantitative PCR detection system was accurate, reliable and sensitive.更多还原