转基因棉花MON757转化体的检测和外源基因表达的初步分析
发布时间：2018-07-31 12:15
【摘要】：外源杀虫蛋白的表达量是转基因抗虫棉安全评价的重要指标。转基因棉花(Gossypium hirsutum)MON757转化体是孟山都公司研发的转cry1Ac基因的抗虫棉,已被美国等批准种植或用于加工原料,但在我国尚未获得批准应用。本实验室前期从我国市场棉花种子中检测到MON757转化体,并进一步繁育筛选出Cry1Ac蛋白表达具有显著差异的转化体材料。本研究以Cry1Ac蛋白表达显著差异的MON757转化体材料为研究对象,建立了转化体特异性的PCR检测方法;初步研究了Cry1Ac蛋白的表达规律,并初步分析了外源片段的整合结构和外源基因的转录水平。获得的主要结果有:一、以转基因棉花MON757转化体的插入位点基因组序列和外源插入片段/基因组连接区序列为靶标,建立了特异性的MON757转化体纯杂合定性PCR检测方法和实时荧光定量PCR检测方法。建立的定性PCR检测方法能准确地鉴别棉花植株和种子中MON757转化体的纯合和杂合状态。建立的定量PCR检测方法(Quantitative Realtime PCR,qRT-PCR)具有良好的可重复性和灵敏度,其检测下限(Limit of Detection,LOD)为11个拷贝,定量下限(Limit of Quantitative,LOQ)为44个拷贝。二、应用酶联免疫吸附测试(enzyme linked immunosorbent assay,ELISA)的方法测定了MON757H型(MON757高表达型)和MON757L型(MON757低表达型)转化体叶片中Cry1Ac蛋白的表达量。结果表明,MON757纯合和杂合类型样品间的叶片中Cry1Ac蛋白表达量没有显著差异,MON757H型叶片中Cry1Ac蛋白表达量维持在较高水平,并随整个生育期的发展呈下降趋势。而MON757L型Cry1Ac蛋白表达量在整个生育时期均保持低水平表达。三、采用普通PCR方法和LD-PCR方法扩增了MON757H型和MON757L型植株外源插入片段的全长序列。结果显示,MON757H型和MON757L型植株外源插入片段结构一致,序列高度相似,表明外源杀虫蛋白表达量的差异不是由外源片段DNA结构引起的。四、运用反转录PCR(reverse transcriptional PCR)方法分析MON757H型和MON757L型外源基因的转录水平。结果表明,MON757H型植株中全长cry1Ac基因和截断的cry1Ac基因的转录水平显著高于MON757L型,而nptⅡ基因的转录水平在两种类型植株中没有显著差异。本研究建立的MON757转化体特异性的PCR检测方法为转基因棉花品种培育和转基因的安全监管提供了重要的技术手段。研究和分析不同类型转基因棉花MON757转化体的外源Cry1Ac蛋白、外源片段整合结构和外源基因的转录,对了解转基因棉花外源基因的表达规律和影响机制以及进行个案化的安全性分析都具有重要的意义。
[Abstract]:The expression of exogenous insecticidal protein is an important index to evaluate the safety of transgenic insect-resistant cotton. Transgenic cotton (Gossypium hirsutum) MON757 transformant, developed by Monsanto Company, has been approved by the United States for planting or processing raw materials, but has not yet been approved for application in China. In our laboratory, MON757 transformants were detected from cotton seeds in our country in early stage, and the transformants with different expression of Cry1Ac protein were further bred and screened. In this study, a specific PCR detection method was established for the detection of MON757 transformants with significant differences in Cry1Ac protein expression, and the expression of Cry1Ac protein was preliminarily studied. The integrative structure of exogenous fragments and the transcription level of exogenous genes were also preliminarily analyzed. The main results are as follows: first, the genomic sequence of insertion site and the sequence of exogenous insert / genomic linkage region of transgenic cotton MON757 transformants were targeted. A specific PCR detection method for pure heterozygosity of MON757 transformants and a real-time fluorescence quantitative PCR detection method were established. The established qualitative PCR detection method can accurately identify the homozygous and heterozygous status of MON757 transformants in cotton plants and seeds. The established quantitative PCR detection method (Quantitative Realtime PCR qRT-PCR has good reproducibility and sensitivity. The detection limit of (Limit of detection lod is 11 copies, and the lower limit of (Limit of quantitative LOQ is 44 copies. Secondly, the expression of Cry1Ac protein in the leaves of MON757H type (MON757 high expression type) and MON757L type (MON757 low expression type) was determined by enzyme-linked immunosorbent assay (Elisa). The results showed that there was no significant difference in the expression of Cry1Ac protein between homozygous and heterozygous samples. The expression of Cry1Ac protein in MON757H type leaves was maintained at a high level and decreased with the development of the whole growth period. However, the expression of MON757L type Cry1Ac protein remained low during the whole growth period. 3. The full-length sequences of exogenous insert fragments of MON757H type and MON757L type plants were amplified by common PCR method and LD-PCR method. The results showed that the structure of exogenous insert fragments was the same as that of MON757L type plants, indicating that the difference in the expression of exogenous insecticidal protein was not caused by the structure of exogenous fragment DNA. Fourthly, reverse transcription PCR (reverse transcriptional PCR) method was used to analyze the transcriptional level of MON757H type and MON757L type foreign genes. The results showed that the transcription level of full-length cry1Ac gene and truncated cry1Ac gene in MON757H type was significantly higher than that in MON757L type, but the transcription level of npt 鈪，
本文编号：2155554
[Abstract]:The expression of exogenous insecticidal protein is an important index to evaluate the safety of transgenic insect-resistant cotton. Transgenic cotton (Gossypium hirsutum) MON757 transformant, developed by Monsanto Company, has been approved by the United States for planting or processing raw materials, but has not yet been approved for application in China. In our laboratory, MON757 transformants were detected from cotton seeds in our country in early stage, and the transformants with different expression of Cry1Ac protein were further bred and screened. In this study, a specific PCR detection method was established for the detection of MON757 transformants with significant differences in Cry1Ac protein expression, and the expression of Cry1Ac protein was preliminarily studied. The integrative structure of exogenous fragments and the transcription level of exogenous genes were also preliminarily analyzed. The main results are as follows: first, the genomic sequence of insertion site and the sequence of exogenous insert / genomic linkage region of transgenic cotton MON757 transformants were targeted. A specific PCR detection method for pure heterozygosity of MON757 transformants and a real-time fluorescence quantitative PCR detection method were established. The established qualitative PCR detection method can accurately identify the homozygous and heterozygous status of MON757 transformants in cotton plants and seeds. The established quantitative PCR detection method (Quantitative Realtime PCR qRT-PCR has good reproducibility and sensitivity. The detection limit of (Limit of detection lod is 11 copies, and the lower limit of (Limit of quantitative LOQ is 44 copies. Secondly, the expression of Cry1Ac protein in the leaves of MON757H type (MON757 high expression type) and MON757L type (MON757 low expression type) was determined by enzyme-linked immunosorbent assay (Elisa). The results showed that there was no significant difference in the expression of Cry1Ac protein between homozygous and heterozygous samples. The expression of Cry1Ac protein in MON757H type leaves was maintained at a high level and decreased with the development of the whole growth period. However, the expression of MON757L type Cry1Ac protein remained low during the whole growth period. 3. The full-length sequences of exogenous insert fragments of MON757H type and MON757L type plants were amplified by common PCR method and LD-PCR method. The results showed that the structure of exogenous insert fragments was the same as that of MON757L type plants, indicating that the difference in the expression of exogenous insecticidal protein was not caused by the structure of exogenous fragment DNA. Fourthly, reverse transcription PCR (reverse transcriptional PCR) method was used to analyze the transcriptional level of MON757H type and MON757L type foreign genes. The results showed that the transcription level of full-length cry1Ac gene and truncated cry1Ac gene in MON757H type was significantly higher than that in MON757L type, but the transcription level of npt 鈪，
本文编号：2155554
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