摘要
随着转基因技术的成熟,转基因生物越来越多,关于转基因生物的争议也越来越受到人们的关注。建立一套能够简便、快速、准确的检测转基因林木的外源基因技术,对转基因林木的研究有重要意义。采用灵敏、快速的环介导等温扩增(loop-mediated isothermal amplification,LAMP)技术,建立一套以Bt Cry1Ac或Npt II基因为靶标的转基因林木的检测方法。针对靶标基因的6个区域设计4种特异引物,优化LAMP-PCR反应体系中的各个成分,根据直接观察产物中焦磷酸镁的沉淀情况、Gold View核酸染色剂对其产物染色情况来判断反应结果并与将产物经过1%琼脂糖电泳的情况进行对比。比较LAMP-PCR和常规PCR两种不同检测方法的灵敏度。对转基因杨树不同无性系中多种外源基因进行检测。筛选出了最佳的引物,确定出了最优的反应体系:3 mmol/L Mg2+、1.4 mmol/L Bst、1.4 mol/L Betaine、7:1的10μmol/L内外引物比、2μL10 mmol/L d NTP、以及2μL浓度为200 ng/μL的DNA模板。在PCR仪65℃等温条件下保温90 min,可直接观察到焦磷酸镁的白色沉淀;经过Gold View核酸染色剂染色,可在紫外灯下观察到荧光绿色;经过1%琼脂糖电泳的检测,可观察到清晰的弥散状条带。通过对比试验得出LAMP-PCR的灵敏度比常规PCR要高。对实验室多个转基因杨树无性系目的基因的检测,证明了LAMP-PCR在转基因林木外源基因检测中的较高可靠性。本研究在国内外首次采用环介导等温扩增技术检测转基因林木,建立了一套更简便、灵敏、快速、可靠的转基因林木的LAMP-PCR检测方法。该技术在恒温65℃的条件下保温90 min即可完成转基因林木的检测,既节省了时间,又降低了成本,为转基因林木的检测研究提供了一种新的技术。
With the development of transgenic technology, the GM（genetically modified） organisms have been paid more and more debate and attention. It is important for the research of transgenic trees to develop a simple,rapid and accurate detection of transgenic trees of exogenous gene technology. A sensitive and rapid loop-mediated isothermal Amplification（LAMP） technique was used to establish the method to test transgenic forest trees with Bt Cry1Ac or Npt II gene as target. Four specific primers were designed in view of six regions of the target genes, to optimize the components of the LAMP-PCR reaction system. The reaction results are determined according to the results of direct observation of precipitation products in magnesium phosphate and staining of Gold View nucleic acid stain on the product. Then the products were compared through the 1% agarose electrophoresis. The sensitivity of two kinds of different detection methods of LAMP-PCR and common PCR were compared, and several exogenous genes were detected in different clones of transgenic poplar by the LAMP-PCR. The best primers were screened out, and the optimal reaction system was determined： 3 mmol/L Mg-（2＋）, 1.4 mmol/L Bst,1.4 mol/L Betaine, 7：1 10 μmol/L inner and outer primers, 2 μL 10 mmol/L d NTP and 2 μL 200 ng/μL DNA.The white precipitate of magnesium pyrophosphate can be directly observed under 65℃ condition for 90 min in the PCR apparatus; by Gold View nucleic acid stain, green fluorescence under can be observed under UV light;then detected by 1% agarose gel electrophoresis and clear diffuse bands can be observed. By contrast, the sensitivity of LAMP-PCR is higher than that of conventional PCR, and the subsequent verification test proves that LAMP-PCR has high reliability in the detection of a variety of target genes in the transgenic poplar clones. This research detected genetically modified trees with the LAMP technology at home and abroad for the first time, and established a simple, rapid, sensitive and reliable LAMP-PCR detection method of genetically modified trees. The technology completing the detection of genetically modified trees under the condition of constant temperature of 65℃ for 90 min that not only saved time but also reduced costs, provided a new technology for the detection of genetically modified trees research.
出处
《分子植物育种》
CAS
CSCD
北大核心
2017年第11期4773-4780,共8页
Molecular Plant Breeding
基金
国家自然科学基金项目(31370663)
国家高技术研究发展计划"(863"计划)项目(2013AA102703)共同资助
关键词
转基因杨树
环介导等温扩增技术
检测
Transgenic poplar, Loop-mediated isothermal amplification （LAMP）, Detection method
