轉基因棉花MON757轉化體的檢測和外源基因表達的初步分析
發(fā)布時間：2018-07-31 12:15
【摘要】：外源殺蟲蛋白的表達量是轉基因抗蟲棉安全評價的重要指標。轉基因棉花(Gossypium hirsutum)MON757轉化體是孟山都公司研發(fā)的轉cry1Ac基因的抗蟲棉,已被美國等批準種植或用于加工原料,但在我國尚未獲得批準應用。本實驗室前期從我國市場棉花種子中檢測到MON757轉化體,并進一步繁育篩選出Cry1Ac蛋白表達具有顯著差異的轉化體材料。本研究以Cry1Ac蛋白表達顯著差異的MON757轉化體材料為研究對象,建立了轉化體特異性的PCR檢測方法;初步研究了Cry1Ac蛋白的表達規(guī)律,并初步分析了外源片段的整合結構和外源基因的轉錄水平。獲得的主要結果有:一、以轉基因棉花MON757轉化體的插入位點基因組序列和外源插入片段/基因組連接區(qū)序列為靶標,建立了特異性的MON757轉化體純雜合定性PCR檢測方法和實時熒光定量PCR檢測方法。建立的定性PCR檢測方法能準確地鑒別棉花植株和種子中MON757轉化體的純合和雜合狀態(tài)。建立的定量PCR檢測方法(Quantitative Realtime PCR,qRT-PCR)具有良好的可重復性和靈敏度,其檢測下限(Limit of Detection,LOD)為11個拷貝,定量下限(Limit of Quantitative,LOQ)為44個拷貝。二、應用酶聯(lián)免疫吸附測試(enzyme linked immunosorbent assay,ELISA)的方法測定了MON757H型(MON757高表達型)和MON757L型(MON757低表達型)轉化體葉片中Cry1Ac蛋白的表達量。結果表明,MON757純合和雜合類型樣品間的葉片中Cry1Ac蛋白表達量沒有顯著差異,MON757H型葉片中Cry1Ac蛋白表達量維持在較高水平,并隨整個生育期的發(fā)展呈下降趨勢。而MON757L型Cry1Ac蛋白表達量在整個生育時期均保持低水平表達。三、采用普通PCR方法和LD-PCR方法擴增了MON757H型和MON757L型植株外源插入片段的全長序列。結果顯示,MON757H型和MON757L型植株外源插入片段結構一致,序列高度相似,表明外源殺蟲蛋白表達量的差異不是由外源片段DNA結構引起的。四、運用反轉錄PCR(reverse transcriptional PCR)方法分析MON757H型和MON757L型外源基因的轉錄水平。結果表明,MON757H型植株中全長cry1Ac基因和截斷的cry1Ac基因的轉錄水平顯著高于MON757L型,而nptⅡ基因的轉錄水平在兩種類型植株中沒有顯著差異。本研究建立的MON757轉化體特異性的PCR檢測方法為轉基因棉花品種培育和轉基因的安全監(jiān)管提供了重要的技術手段。研究和分析不同類型轉基因棉花MON757轉化體的外源Cry1Ac蛋白、外源片段整合結構和外源基因的轉錄,對了解轉基因棉花外源基因的表達規(guī)律和影響機制以及進行個案化的安全性分析都具有重要的意義。
[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，
本文編號：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，
本文編號：2155554
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