转2mG2-epsps基因抗草甘膦玉米品系分子特征及特异性检测
发布时间：2018-08-24 17:03
【摘要】：转基因玉米是最早获得商业化种植的转基因作物之一,也是世界上种植面积最大的转基因经济、粮食作物。我国至今尚无转基因玉米品系获批产业化生产,原因之一就是转基因玉米的分子特征信息不完善,阻碍了相应品系的安全评价工作。G2-epsps是本研究室自主克隆的抗草甘膦基因,根据植物密码子偏好性对G2-epsps进行优化,获得新型抗草甘膦基因2m G2-epsps。新型转2m G2-epsps基因玉米在田间试验中表现出良好的抗草甘膦特性。本研究以转2m G2-epsps基因玉米品系为研究对象,利用反转录(RT-PCR)和高效热不对称交互PCR(hi TAIL-PCR)技术及分子杂交技术,分析了插入外源T-DNA的拷贝数及侧翼序列,建立了相应玉米新品系特异性检测方法。本研究获得了转基因玉米品系的分子特征信息、检测方法,为申请安全证书以及商业化后的检测、监管提供有力的保障。主要研究结果如下:(1)利用反转录PCR和Western Bolt分析转基因玉米品系D、T255、T254、T257中外源基因2m G2-epsps的表达。结果表明,外源基因2m G2-epsps在转基因玉米品系D、T255、T254、T257中正常转录与表达,与本实验室自主研发的抗除草剂G2-EPSPS快速检测试纸条检测的结果一致。(2)通过Southern Blot、微滴式数字PCR(Droplet Digital PCR)测定转基因玉米品系D、T255、T254、T257插入外源基因T-DNA拷贝数。结果显示,转基因玉米品系D的外源基因拷贝数为2个,品系T254、T257外源基因拷贝数约为3,品系T255外源基因拷贝数约为4。因此将转基因玉米品系D作为下一步研究的材料。(3)通过3'端1次hi TAIL-PCR和1次长链PCR扩增方法获得了转抗草甘膦基因2m G2-epsps玉米品系D的外源DNA插入片段的全DNA序列(T-DNA)及两端侧翼序列。发现外源插入序列共3827 bp,其中包括一个完整拷贝的2m G2-epsps基因序列,以及一个玉米泛素化启动子ubiquitin promoter、一个完整终止子Nos-Ter,并且外源基因2m G2-epsps的5'端还带有叶绿体导肽CTP。外源T-DNA的第一个插入位点位于玉米基因组的Ⅰ号染色体上(227622408..227656679),第二个插入位点位于位于玉米基因组的Ⅱ号染色体上(113077961-113077617)。(4)转基因玉米D品系特异性检测方法的建立。根据获得的外源T-DNA序列与玉米基因组的连接区即侧翼序列设计PCR检测方法的引物,建立了D品系特异性普通PCR检测方法,以非转基因玉米、油菜、水稻、大豆和转基因玉米、油菜、水稻、大豆为材料,验证该方法的特异性及灵敏度,最低检测限约为0.05%。
[Abstract]:Transgenic corn is one of the first transgenic crops to be grown commercially, and it is also the largest genetically modified economy in the world. Up to now, no transgenic maize lines have been approved for industrial production in China. One of the reasons is that the molecular characteristic information of transgenic maize is not perfect, which hinders the safety evaluation of the corresponding lines. G2-epsps is the glyphosate resistant gene cloned independently in our laboratory. According to plant codon preference, G2-epsps was optimized and a novel glyphosate resistant gene 2m G2-epsps was obtained. The transgenic 2m G2-epsps maize showed good glyphosate resistance in field trials. In this study, the copy number and flanking sequence of exogenous T-DNA were analyzed by means of reverse transcription (RT-PCR), highly efficient thermal asymmetric interactive PCR (hi TAIL-PCR (PCR (hi TAIL-PCR) and molecular hybridization. The specific detection method of the new maize strain was established. In this study, the molecular characteristic information and detection methods of transgenic maize lines were obtained, which provided a strong guarantee for the application for safety certificates and for commercial detection and supervision. The main results were as follows: (1) reverse transcription PCR and Western Bolt were used to analyze the expression of 2m G2-epsps gene in transgenic maize strain DG T255N T254N T257. The results showed that the foreign gene 2m G2-epsps was normally transcribed and expressed in transgenic maize strain DG255T254T254T257. The results were consistent with the results of G2-EPSPS rapid test strip developed by our laboratory. (2) the copy number of T-DNA inserted into transgenic maize strain DT255T254T257 was determined by Southern Blot, microdrop digital PCR (Droplet Digital PCR). The results showed that the foreign gene copy number of transgenic maize strain D was 2, the copy number of foreign gene of strain T254N T257 was about 3, and the copy number of foreign gene of strain T255 was about 4. Therefore, transgenic maize strain D was used as the next material for further study. (3) the whole DNA sequence of exogenous DNA insert fragment of glyphosate resistant 2m G2-epsps maize strain D was obtained by 3 'terminal hi TAIL-PCR amplification and one long chain PCR amplification method. (T-DNA) and two flanking sequences. A total of 3827 bp, was found, including a complete copy of the 2m G2-epsps gene sequence, a maize ubiquitin promoter ubiquitin promoter, a complete Terminator Nos-Ter, and the 5'terminal of the foreign gene 2m G2-epsps with a chloroplast conducting peptide CTP.. The first insertion site of exogenous T-DNA was on chromosome I of maize genome (227622408..227656679), and the second was located on chromosome 鈪，
本文编号：2201455
[Abstract]:Transgenic corn is one of the first transgenic crops to be grown commercially, and it is also the largest genetically modified economy in the world. Up to now, no transgenic maize lines have been approved for industrial production in China. One of the reasons is that the molecular characteristic information of transgenic maize is not perfect, which hinders the safety evaluation of the corresponding lines. G2-epsps is the glyphosate resistant gene cloned independently in our laboratory. According to plant codon preference, G2-epsps was optimized and a novel glyphosate resistant gene 2m G2-epsps was obtained. The transgenic 2m G2-epsps maize showed good glyphosate resistance in field trials. In this study, the copy number and flanking sequence of exogenous T-DNA were analyzed by means of reverse transcription (RT-PCR), highly efficient thermal asymmetric interactive PCR (hi TAIL-PCR (PCR (hi TAIL-PCR) and molecular hybridization. The specific detection method of the new maize strain was established. In this study, the molecular characteristic information and detection methods of transgenic maize lines were obtained, which provided a strong guarantee for the application for safety certificates and for commercial detection and supervision. The main results were as follows: (1) reverse transcription PCR and Western Bolt were used to analyze the expression of 2m G2-epsps gene in transgenic maize strain DG T255N T254N T257. The results showed that the foreign gene 2m G2-epsps was normally transcribed and expressed in transgenic maize strain DG255T254T254T257. The results were consistent with the results of G2-EPSPS rapid test strip developed by our laboratory. (2) the copy number of T-DNA inserted into transgenic maize strain DT255T254T257 was determined by Southern Blot, microdrop digital PCR (Droplet Digital PCR). The results showed that the foreign gene copy number of transgenic maize strain D was 2, the copy number of foreign gene of strain T254N T257 was about 3, and the copy number of foreign gene of strain T255 was about 4. Therefore, transgenic maize strain D was used as the next material for further study. (3) the whole DNA sequence of exogenous DNA insert fragment of glyphosate resistant 2m G2-epsps maize strain D was obtained by 3 'terminal hi TAIL-PCR amplification and one long chain PCR amplification method. (T-DNA) and two flanking sequences. A total of 3827 bp, was found, including a complete copy of the 2m G2-epsps gene sequence, a maize ubiquitin promoter ubiquitin promoter, a complete Terminator Nos-Ter, and the 5'terminal of the foreign gene 2m G2-epsps with a chloroplast conducting peptide CTP.. The first insertion site of exogenous T-DNA was on chromosome I of maize genome (227622408..227656679), and the second was located on chromosome 鈪，
本文编号：2201455
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