建立转基因作物及产品外源抗性基因检测技术体系的研究

【作者】 曹际娟；

【导师】 曹远银；

【作者基本信息】 沈阳农业大学 ， 植物病理学， 2004， 博士

【摘要】 自1983年世界首例转基因烟草作物问世以来，转基因作物作为一类高经济效益的作物以无与伦比的速度迅速应用，但转基因作物在生态和食品安全方面的问题仍不清楚，在知识产权和产品加贴标签问题等方面的争议也非常巨大。因此，转基因作物的检测问题亟待解决。 本文全面收集整理了目前所有的16类作物、73个品系抗性基因的质粒图谱信息资料，以这些信息资料为依据，展开了DNA分子前沿检测技术改进与应用研究：采用反向PCR克隆—测序、修饰基因破译方法，获得品系鉴定边界序列和修饰基因序列，并设计特异性引物对特定区域进行常规PCR扩增(定性检测)、实时荧光PCR扩增(定性／定量检测)和高通量基因芯片检测技术(多品系多种类作物检测)的系统研究，并把这些技术首次应用于转基因作物及其产品的外源基因鉴定检测，获得巨大成功。本研究首次系统地建立了国内外第一个转基因作物及产品外源抗性基因检测技术体系，这对于我国加入WTO后检测进出口转基因作物及其产品、加贴转基因标签、消除发达国家农作物与产品贸易壁垒，仲裁国际农产品安全问题争端都就具有至关重要的意义。 本研究采用反向PCR克隆—测序测定未知序列技术，针对转基因玉米(MON810、BT11、BT176、GA21、T25、CBH-351)六个品系、转基因大豆GTS 40-3-2品系、转基因油菜RT73和MS8两个品系、转基因棉花MON531和MON1445两个品系测定出品系鉴定的边界序列；采用外源修饰基因序列的破译方法和BLAST同源性序列分析技术，针对转基因作物中修饰的外源抗性基因进行破译研究，破译出转基因油菜中修饰的GOX基因、修饰的CP4-EPSPS基因和转基因棉花中修饰的CrylA(c)基因的序列，并确定了转基因马铃薯New leaf(?)PLUS和New leaf(?)Y两个品系中PLRVrep、PVYcp和CryⅢA外源抗性基因保守序列。根据基因序列，设计并筛选优化适合于常规PCR检测的特异性引物，建立了转基因作物19个外源抗性基因的常规 摘要PCR检测技术。根据品系鉴定的边界序列和品系特异的抗性基因，设计并筛选优化适合于实时荧光PCR检测的特异性引物和探针，并通过研究比较TaqMan探针和sYB脉Green实时荧光PcR方法，以及系统研究比较转基因作物的三种定量检测方法，从而建立了转基因作物具抗性功能的n个品系的实时荧光PCR鉴定技术和转基因作物基于标准分子质粒DNA的定量检测新技术。在基因芯片检测研究中，优化出反应体系和反应条件几乎一致的29条引物和29条探针，并使多重PCR能做到10重引物的同时扩增，克服了多重PCR反应条件优化过程中各引物之间的相互干扰，各探针与多重PCR反应产物之间的相互干扰，以及引物、探针、荧光标记产物的长度和结构之间等存在的相互干扰等技术难点，并经研究比较确定寡核昔酸芯片在转基因作物及产品检测方面优于cDNA芯片;同时根据转基因作物中常见的外源抗性基因、物种特异基因和品系鉴定边界序列的特点，研制出转基因作物及产品的筛选检测芯片、物种结构基因检测芯片和品系鉴定芯片，以及集犯个基因于一体的综合型检测芯片，从而建立了基于多重PCR技术的高通量检测转基因作物及产品的基因芯片检测技术。 通过对研究建立的作物内源参照的玉米zssllb基因、大豆Lectin基因、油菜FatA基因、棉花ACPI基因和马铃薯Patatin基因的检测，使本研究的检测体系得到了质量控制的保障。通过与国际上88个实验室间转基因定性和定量检测的比对测试研究，客观地证明了本研究建立的转基因作物及产品外源抗性基因检测技术体系的有效性和完善性。 本研究首次建立了转基因作物及产品外源抗性基因的常规PCR、实时荧光PCR和基因芯片检测以及国际实验室间比对试验验证的技术体系，这也是国际上首次研究建立的转基因作物及产品外源抗性基因检测技术体系。更多还原

【Abstract】 Since the first genetically modified crop tobacco came into being in the word in 1983, the genetically modified crops as a cluster of highest economic return crops have been widely spread in an incomparable speed throughout the globe, however, the ecological risk, food safety anxiety, intellectual property rights and product tagging etc. have been on debate. Thus, the detection for genetically modified crops is urgently needed.In this paper, 73 lines in 16 kinds of resistance gene plasmid schemes contained in the genetically modified crops were collected and classified. Based on the information, the researches on advanced DNA molecular detection approaches were carried out: by using reverse PCR cloning-sequencing modified gene decoding to obtain differentiating flanking gene and modified gene sequence and modified gene sequences, designing specific primer to amplify specific region by conventional PCR(qualitative detection) and by real time PCR (qualitative and quantitative detections) and high density genetic chip multi-crop line detection, those newly developed detection protocols were for the first time in the world utilized to detect exogenous resistance genes in genetically modified crops and products, which have momentous meanings for detecting exogenous resistant genes in imported and exported GMO and products, tagging GMO and products, removing farm crop product trade barriers and drawbacks imposed by developed nations and judging international disputes on agricultural crops and products in accordance with principle of WTO.Crop line differentiating flanking sequences of 6 lines of genetically modified (GM) maize (MON810, BT11, BT176, GA21, T25 and CBH-351) and 1 line of GM soybean GTS 40-3-2, 2 lines of GM canola (RT73 and MS8) and 2 lines of GM cotton (MON531 and MON1445) were known using inverted PCR cloning-sequencing approach to determine unknown sequences. Sequences of modified genes GOX and CP4-EPSPS in GM canola and that of modified gene Cryla(c) in GM cotton were decoded and the conservative sequences of exogenous resistant genes : PLRVrep, PVYcp and CryIIIA in GM potato: New leaf?PLUS and New leaf?Y were confirmed using the modified exogenous gene decoding technique and isogenous sequence similarity BLAST analysis. Bases on DNA sequences studied above, conventional PCR primers were designed and selected in an optimized way and the conventional PCR detection protocol for exogenous resistant genes in 19 GM crops was established. Based on crop line differentiating flanking sequences and crop line specific resistant genes in GMO, specific primers and probes suitable for real time PCR detection were designed and selected.Different protocols for real time PCR with probe Taqman and fluorescein SYBR瓽reen and three quantitative detection methods for GMO were systematically compared, from these studies, the real time PCR detection protocol for 11 resistance genes and the quantitative protocol based on standard molecular plasmids in GMO were established.In the study of genetic chip of resistance gene detection 29 primers and 29 probes which are uniform in PCR detection profile and condition were selected and optimized, which could allow 10 pairs of primers in one multiple PCR reaction to perform amplifying reaction simultaneously, so the interaction interferences between primer pairs in the process of multiple PCR reaction condition optimization , between every pair of primers and products of multiple PCR reaction and between oligonucleotide length and structure of primers, probes and fluorescein labeled products were overcome. The studies shown above clearly indicated that oligonucleotide chip was better than cDNA chip in consideration of exogenous gene detection in GMO. Further more, taking the advantage of the special traits of common exogenous resistance genes, species specific genes and crop line differentiating Flanking sequences, the GMO and product sieving detection chip, species structural gene detection chip, crop line differentiating chip and 32 gene integrated detection chip were更多还原