转基因作物分子检测方法开发及转AgGlpF基因耐盐大豆材料创制

发布时间：2018-04-13 03:26

  本文选题：转基因作物 + 环介导等温扩增　； 参考：《吉林大学》2017年博士论文

【摘要】：本研究根据我国转基因生物安全监管对快速、精准、高通量检测技术的需求,以转基因作物中常见外源抗虫基因及新研发的转基因玉米转化体为靶标,基于普通PCR、定量PCR、数字PCR、多重PCR、LAMP等技术,旨在建立相应转基因成分的核酸分子检测方法。本研究还利用基因工程手段将前期从嗜盐曲霉中克隆、并已在拟南芥中证实具有耐盐潜力的AgGlp F基因转入大豆栽培品种,以创制耐盐转基因大豆新材料,为耐盐大豆新品种培育提供种质资源。主要研究结果如下:1.为建立转基因作物中抗虫基因的筛选检测方法,我们系统分析了国内外转基因作物中常用的外源抗虫基因种类及其核苷酸序列,并根据序列一致性程度,将cry1Ab、cry1Ac、cry1Ab/Ac、mcry1Ac、cry1A.105、cry2Ab、cry3A、cry3Bb等8种Bt基因分为Cry1A、Cry2A和Cry3A等3个类别,分别建立了这3类Bt基因的单一PCR检测方法及通过一次扩增同时检测这3类基因的三重PCR方法,其中Cry1A类基因检测方法采用简并PCR策略。上述方法具有特异性强、灵敏度高等特点,检测灵敏度均可达0.1%,非常适用于转基因作物中cry1Ab、cry1Ac、cry1Ab/Ac等常见Bt基因的快速高效检测。2.除了常规PCR方法,本研究还开展了转基因作物中cry2Ab和cry3A基因LAMP快速检测方法研究。结果表明,应用这两种LAMP方法可特异性地将含有相应靶标基因的转基因作物与其他转基因作物和非转基因作物鉴别开,方法的检出限均可达5个靶标DNA序列拷贝,比常规PCR方法的灵敏度提高了4倍。而且,应用LAMP方法可在1个小时内完成检测,比常规PCR方法用时缩短一半以上。通过在LAMP反应体系中加入荧光染料,还能够实现肉眼可视化检测,为转基因成分的现场快速检测提供了一种可靠的技术手段。3.为加强我国自主研发的转基因玉米IE034和Bt506的安全评价、安全监管及知识产权保护,本研究开展了这两种转基因玉米的转化体特异性定性及定量PCR精准检测方法研究,包括特异性、灵敏度、检出限、定量限、准确度和精确度测试等。结果表明,IE034和Bt506玉米的转化体特异性PCR方法对相应转化体具有严格的专一性,这两种转基因玉米的定性PCR方法检测灵敏度均可达0.05%,相当于约20个基因组DNA拷贝。IE034玉米定量PCR方法的检测限及定量限分别为8个和40个拷贝,对IE034玉米的转化体质量分数分别为5%、1%、0.5%和0.23%的4个实际样品的定量测定值与预期值之间的偏差分别为12.2%、3.0%、8.0%和8.7%。Bt506玉米定量PCR方法的检测限及定量限分别为4个和35个拷贝,对Bt506玉米的转化体质量分数分别为5%、2%和0.5%的3个实际样品的定量测定值与预期值之间的偏差分别为10.96%、18.08%和12.64%,均符合ENGL发布并被国内外同行广泛认可的评价标准。综上所述,本研究建立的检测方法可对IE034及Bt506转化体成分进行精确的定性鉴定和定量分析。4.建立了我国自主研发的转基因玉米Bt38的荧光定量PCR方法(q PCR)。应用该方法对2个实际样品的鉴定结果表明,无论是单重还是双重定量PCR体系,均能获得准确可靠的定值结果。利用单重q PCR方法获得的测定值与预期值之间的偏差分别为7.44%和14.28%,利用双重q PCR方法定值的偏差分别为2.50%和17.34%。此外,我们还成功将Bt38玉米的q PCR方法转化为微滴式数字PCR方法(dd PCR)。应用Bt38玉米的单重和双重dd PCR方法,对上述2个实际样品的定量结果均与q PCR结果一致,表明建立的Bt38玉米q PCR和dd PCR方法均适合于Bt38转化体成分的身份鉴定及精准定量分析。5.利用农杆菌介导的大豆子叶节转化法,将AgGlp F基因转入大豆受体材料Willams82中,经除草剂筛选及PCR、Southern杂交鉴定,获得29株转AgGlp F基因大豆阳性植株,并从T1代植株中筛选出AgGlp F基因以单拷贝形式整合到大豆基因组中的3个转基因大豆品系。反转录荧光定量PCR结果表明,AgGlp F基因在转基因材料E8A7027-816和E8A7027-817的叶片、根、茎等不同组织器官中均能稳定转录表达,其中叶片的表达量最高,其次为茎、根。转基因大豆材料E8A7027-817在200mmol/L的Na Cl盐胁迫下能够正常生长,而非转基因大豆对照材料在盐胁迫处理3周后枯萎死亡,表明过表达AgGlp F基因能显著提高大豆的耐盐性,这为获得耐盐转基因大豆提供了一种有效的途径。此外,建立了AgGlp F基因特异性的荧光定量PCR方法,为转AgGlp F基因大豆后代材料鉴定及基因表达量分析提供方法支持。
[Abstract]:On the basis of GMO Safety Supervision in China on the fast, accurate, high-throughput detection technology needs to genetically modified crops in common exogenous insect resistant genes and transgenic maize new transformants as the target, based on common PCR, quantitative PCR, digital PCR, multiplex PCR, LAMP technology, to establish the corresponding detection method of nucleic acid molecules genetically modified ingredients. This study also used genetic engineering means to pre from halophilic Aspergillus clones, and confirmed with salt tolerance potential of AgGlp F gene into soybean cultivars in Arabidopsis, to create salt tolerant transgenic soybean germplasm resources to provide new materials, new varieties of salt tolerant soybean cultivation. The main results are as follows 1.: to establish a method of detecting for the screening of transgenic crop gene, we analyzed the domestic and foreign GM crops commonly used in insect resistant gene types and exogenous nucleotide sequences, According to the sequence and the degree of consistency, cry1Ab, cry1Ac, cry1Ab/Ac, mcry1Ac, cry1A.105, cry2Ab, cry3A, cry3Bb and other 8 kinds of Bt gene into Cry1A, Cry2A and Cry3A 3 categories, a single PCR detection method of the 3 kinds of Bt gene were established and amplified through a three PCR method at the same time. Measurement of these 3 genes, including Cry1A gene detection method using degenerate PCR strategy. The method has strong specificity, high sensitivity, the detection sensitivity can reach 0.1%, so it is very suitable for cry1Ab, cry1Ac and cry1Ab/Ac in transgenic crops, because of the common Bt based fast and efficient detection of.2. PCR in addition to conventional methods, this study also carried out cry2Ab and cry3A genes in transgenic crops LAMP method for rapid detection of genetically modified crops. The results showed that the application of the two kinds of LAMP methods specifically will contain the target gene with other transgenic and non transgenic crops Crop identification, the detection limit can reach 5 copies of target DNA sequence, sensitivity than the conventional PCR method is increased by 4 times. Moreover, the application of LAMP detection method can be completed in 1 hours, compared to the conventional PCR method for reduction of more than half. The fluorescent dye is added in the LAMP reaction system, but also can the naked eye visual detection, scene for the rapid detection of genetically modified components provides a reliable technical means to strengthen the safety evaluation.3. China's independent research and development of transgenic maize IE034 and Bt506, safety supervision and protection of intellectual property rights, this paper carried out qualitative and quantitative conversion of specific PCR these two kinds of genetically modified corn precision detection research methods, including specificity, sensitivity, detection limit, limit of quantification, accuracy and precision of testing. The results showed that the transformant specific PCR method of IE034 and Bt506 in maize transformation of corresponding body. There are strict specificity, qualitative PCR method of these two kinds of genetically modified corn detection sensitivity can reach 0.05%, the detection limit and the limit of quantification is equivalent to approximately 20 genome copies of DNA quantitative PCR method.IE034 maize were 8 and 40 copies, the mass fraction of IE034 maize transformants respectively 5%, 1%. Quantitative 4 actual samples 0.5% and 0.23% of the measured value and the expected difference between the value were 12.2%, 3%, 8% and the LOD and LOQ of 8.7%.Bt506 corn quantitative PCR method were 4 and 35 copies, the mass fraction of transformants of Bt506 corn were 5%, 3 and 2% quantitative samples and 0.5% of the measured value and the expected difference between the value were 10.96%, 18.08% and 12.64%, are in line with ENGL issued by the domestic and foreign colleagues and widely accepted evaluation criteria. In summary, this study established the detection method of composition of IE034 and Bt506 transformed body Qualitative identification and quantitative analysis of the precise.4. method was established for fluorescence quantitative PCR transgenic maize Bt38 developed in China (Q PCR). The identification results by this method in 2 real samples showed that both single and double quantitative PCR system can obtain accurate and reliable results of fixed value. Using the single Q method for the determination of PCR value and the expected value of the deviation between 7.44% and 14.28% respectively, the deviation value by the double Q PCR method were 2.50% and 17.34%. respectively. In addition, we also successfully Q PCR method of Bt38 corn into the droplet digital PCR (DD PCR). The application of Bt38 in Maize Single and double DD PCR method, the quantitative of the 2 sample results and Q PCR results showed that Bt38, PCR and DD PCR of maize Q methods were suitable for conversion of Bt38 body composition identification and accurate quantitative analysis of.5. using Agroinoculation Cotyledonary node transformation mediated by bacteria, F AgGlp gene into soybean receptor in Willams82 by herbicide selection and PCR Southern hybridization, 29 strains of transgenic AgGlp soybean F gene positive plants, and from the T1 generation plants screened F gene AgGlp in the form of single copy integration to 3 GM soybean lines in the soybean genome. Reverse transcription fluorescence quantitative PCR results showed that the leaf, the F gene AgGlp in transgenic materials E8A7027-816 and E8A7027-817 of the root, stem in different organs are stable expression, which leaves the expression level was the highest, followed by stems and roots. The transgenic soybean material E8A7027-817 can grow normally in 200mmol/L Na Cl under salt stress, while the non transgenic soybean control materials in salt treated die after 3 weeks, showed that over expression of F gene of AgGlp can significantly improve the salt tolerance of soybean, which in order to obtain transgenic salt tolerance Soybean provides an effective way. In addition, a fluorescence quantitative PCR method for AgGlp F gene is established, which provides support for material identification and gene expression analysis of AgGlp F transgenic soybean progeny.

【学位授予单位】：吉林大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：S565.1
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本文编号：1742722