【摘要】：轉(zhuǎn)基因玉米是最早獲得商業(yè)化種植的轉(zhuǎn)基因作物之一,也是世界上種植面積最大的轉(zhuǎn)基因經(jīng)濟(jì)、糧食作物。我國(guó)至今尚無(wú)轉(zhuǎn)基因玉米品系獲批產(chǎn)業(yè)化生產(chǎn),原因之一就是轉(zhuǎn)基因玉米的分子特征信息不完善,阻礙了相應(yīng)品系的安全評(píng)價(jià)工作。G2-epsps是本研究室自主克隆的抗草甘膦基因,根據(jù)植物密碼子偏好性對(duì)G2-epsps進(jìn)行優(yōu)化,獲得新型抗草甘膦基因2m G2-epsps。新型轉(zhuǎn)2m G2-epsps基因玉米在田間試驗(yàn)中表現(xiàn)出良好的抗草甘膦特性。本研究以轉(zhuǎn)2m G2-epsps基因玉米品系為研究對(duì)象,利用反轉(zhuǎn)錄(RT-PCR)和高效熱不對(duì)稱交互PCR(hi TAIL-PCR)技術(shù)及分子雜交技術(shù),分析了插入外源T-DNA的拷貝數(shù)及側(cè)翼序列,建立了相應(yīng)玉米新品系特異性檢測(cè)方法。本研究獲得了轉(zhuǎn)基因玉米品系的分子特征信息、檢測(cè)方法,為申請(qǐng)安全證書(shū)以及商業(yè)化后的檢測(cè)、監(jiān)管提供有力的保障。主要研究結(jié)果如下:(1)利用反轉(zhuǎn)錄PCR和Western Bolt分析轉(zhuǎn)基因玉米品系D、T255、T254、T257中外源基因2m G2-epsps的表達(dá)。結(jié)果表明,外源基因2m G2-epsps在轉(zhuǎn)基因玉米品系D、T255、T254、T257中正常轉(zhuǎn)錄與表達(dá),與本實(shí)驗(yàn)室自主研發(fā)的抗除草劑G2-EPSPS快速檢測(cè)試紙條檢測(cè)的結(jié)果一致。(2)通過(guò)Southern Blot、微滴式數(shù)字PCR(Droplet Digital PCR)測(cè)定轉(zhuǎn)基因玉米品系D、T255、T254、T257插入外源基因T-DNA拷貝數(shù)。結(jié)果顯示,轉(zhuǎn)基因玉米品系D的外源基因拷貝數(shù)為2個(gè),品系T254、T257外源基因拷貝數(shù)約為3,品系T255外源基因拷貝數(shù)約為4。因此將轉(zhuǎn)基因玉米品系D作為下一步研究的材料。(3)通過(guò)3'端1次hi TAIL-PCR和1次長(zhǎng)鏈PCR擴(kuò)增方法獲得了轉(zhuǎn)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)及兩端側(cè)翼序列。發(fā)現(xiàn)外源插入序列共3827 bp,其中包括一個(gè)完整拷貝的2m G2-epsps基因序列,以及一個(gè)玉米泛素化啟動(dòng)子ubiquitin promoter、一個(gè)完整終止子Nos-Ter,并且外源基因2m G2-epsps的5'端還帶有葉綠體導(dǎo)肽CTP。外源T-DNA的第一個(gè)插入位點(diǎn)位于玉米基因組的Ⅰ號(hào)染色體上(227622408..227656679),第二個(gè)插入位點(diǎn)位于位于玉米基因組的Ⅱ號(hào)染色體上(113077961-113077617)。(4)轉(zhuǎn)基因玉米D品系特異性檢測(cè)方法的建立。根據(jù)獲得的外源T-DNA序列與玉米基因組的連接區(qū)即側(cè)翼序列設(shè)計(jì)PCR檢測(cè)方法的引物,建立了D品系特異性普通PCR檢測(cè)方法,以非轉(zhuǎn)基因玉米、油菜、水稻、大豆和轉(zhuǎn)基因玉米、油菜、水稻、大豆為材料,驗(yàn)證該方法的特異性及靈敏度,最低檢測(cè)限約為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，

本文編號(hào)：2201455