转基因抗虫棉安全性评价及分子生物学检测体系建立的研究
摘要
国际上转基因作物大规模发展，目前我国已经产业化的转基因作物主要是抗虫棉。抗虫棉的应用和推广为棉花生产带来了巨大的社会和经济效益，但近年来随着转基因作物安全性问题的提出，转基因棉花的安全性问题越来越多地引起了人们的关注。我国转基因农作物的安全性评价体系已经建立，并日趋完善。目前所系统申报的主要是转基因抗虫棉品种。本研究针对我国转基因农作物安全性申报的要求，建立了转基因抗虫棉的分子生物学检测体系，是我国转基因抗虫棉产业化的基础性研究，为转基因植物的分子检测提供了一定依据。研究结果如下：
1）提出了一种新的提取棉花总DNA的方法——SDS结合CTAB法，有效克服了棉花富含酚、多糖等次生代谢物的困难，使所得棉花DNA纯度高，OD260/OD280达到1.8，并提高了DNA产量，达到15ng/mg鲜叶。
2）建立了快速鉴定中、美抗虫棉的PCR体系及反应条件，确定模板DNA的最佳量为50ng，最佳反应条件为降落PCR：
94℃ 预变性7min
94℃ 45sec, 65℃退火45sec, 72℃延伸1min, 重复2个循环；
94℃ 45sec, 63℃退火45sec, 72℃延伸1min, 重复2个循环；
94℃ 45sec, 61℃退火45sec, 72℃延伸1min，重复2个循环；
94℃ 45sec, 59℃退火45sec, 72℃延伸1min，重复2个循环；
94℃ 45sec, 57℃退火45sec, 72℃延伸1min，重复2个循环；
94℃ 45sec, 55℃退火45sec, 72℃延伸1min，重复2个循环；
94℃ 45sec, 52℃退火45sec, 72℃延伸1min，重复24个循环；
72℃保温7min。
3）摸索出了DIG Southern杂交检测GFM cry1A 基因在抗虫棉基因组中整合拷贝数的方法，确定棉花基因组用量为30μg。通过Southern杂交检测到Bt杀虫基因在8个单价转基因抗虫棉和5个双价转基因抗虫棉基因组中整合的拷贝数为1至4。
4）通过检测抗虫棉种植区内其它植物基因组中无外源GFM cry1A基因及cpti基因，证明转基因抗虫棉的外源基因没有向棉花种植地的其它植物中漂移。
5）尝试了用专用试纸条对抗虫棉杀虫蛋白表达的初步测定，检测到Cry1A蛋白在13个抗虫棉品种（系）中得到不同程度的表达。
In recent years, the culture of GMC(genetically modified crops) has developed on a large scale all over the world and the main Chinese GMC turned into industrialization is insect-resistant cotton, which has bought great benefit to the society, but the safety of the transgene arouse more and more attention.
The system of GMC identification has been established in China and now we report a insect-resistant cotton identifying system according to the transgene safety application of China at the molecular level. This is a basis reaserch of insect-resistant trangenetic cotton industrialization and our research may benefit to the molecular identification of GMC in china. The results are as below:
1.A modified method for extracting total DNA from cotton has been accquired. This method can eliminate the disturbance of polysaccharide and polyphenol which is very rich in cotton effectively and the total DNA obtained by this method showed to be more integral and pure. The ratio of OD260 and OD280 is about 1.8 and the yield is about 15ng DNA per milligram tissue.
2.We have established a PCR system which can rapidly identify Chinses insect-resistant cotton and America insect-resistant cotton. The optimal amount of the template is 50ng and the suitable condition is touch-down PCR:
preequilibrated the PCR system to the denaturation temperature 94℃ for 7min and then,
94℃ denaturation for 45sec, 63℃ annealing of primer for 45sec, 72℃primer extension for 1min, 2 cycles；
94℃ denaturation for 45sec, 61℃ annealing of primer for 45sec, 72℃primer extension for 1min, 2 cycles；
94℃ denaturation for 45sec, 59℃ annealing of primer for 45sec, 72℃primer extension for 1min, 2 cycles；
94℃ denaturation for 45sec, 57℃ annealing of primer for 45sec, 72℃primer extension for 1min, 2 cycles；
94℃ denaturation for 45sec, 55℃ annealing of primer for 45sec, 72℃primer extension for 1min, 2 cycles；
94℃ denaturation for 45sec, 52℃ annealing of primer for 45sec, 72℃primer extension for 1min, 2 cycles；
Followed by:Final extension:72℃ for 7min
3.the the number of GFM cry1A copy in insect-resistant cotton genome has been detection
by the method of Southern blotting and we suggest the optimal amount of genomic DNA for Southern is 30μg. The southern blotting show Bt toxin gene in 8 insect-resistant trangenetic cotton harbing single gene lines and 和5 insect-resistant trangenetic cotton harbing double gene lines is 1 to 4 copies respectively.
4.The detection of GFM cry1A gene and cpti show the gene flow has not accur between insect-resistant transgenic cotton and other plant in the regions of insect-resistant transgenic cotton.
5. we have identify the expression of GFM cry1A gene at the protein level by special test strip and the result show GFM cry1A gene express differently in 13 insect-resistant trangenetic cotton lines.
Postgraduate: Sun Xin Majo: Botany
Supervisor: Prof. Hu Baozhong Prof. Guo Sandui
1）提出了一种新的提取棉花总DNA的方法——SDS结合CTAB法，有效克服了棉花富含酚、多糖等次生代谢物的困难，使所得棉花DNA纯度高，OD260/OD280达到1.8，并提高了DNA产量，达到15ng/mg鲜叶。
2）建立了快速鉴定中、美抗虫棉的PCR体系及反应条件，确定模板DNA的最佳量为50ng，最佳反应条件为降落PCR：
94℃ 预变性7min
94℃ 45sec, 65℃退火45sec, 72℃延伸1min, 重复2个循环；
94℃ 45sec, 63℃退火45sec, 72℃延伸1min, 重复2个循环；
94℃ 45sec, 61℃退火45sec, 72℃延伸1min，重复2个循环；
94℃ 45sec, 59℃退火45sec, 72℃延伸1min，重复2个循环；
94℃ 45sec, 57℃退火45sec, 72℃延伸1min，重复2个循环；
94℃ 45sec, 55℃退火45sec, 72℃延伸1min，重复2个循环；
94℃ 45sec, 52℃退火45sec, 72℃延伸1min，重复24个循环；
72℃保温7min。
3）摸索出了DIG Southern杂交检测GFM cry1A 基因在抗虫棉基因组中整合拷贝数的方法，确定棉花基因组用量为30μg。通过Southern杂交检测到Bt杀虫基因在8个单价转基因抗虫棉和5个双价转基因抗虫棉基因组中整合的拷贝数为1至4。
4）通过检测抗虫棉种植区内其它植物基因组中无外源GFM cry1A基因及cpti基因，证明转基因抗虫棉的外源基因没有向棉花种植地的其它植物中漂移。
5）尝试了用专用试纸条对抗虫棉杀虫蛋白表达的初步测定，检测到Cry1A蛋白在13个抗虫棉品种（系）中得到不同程度的表达。
In recent years, the culture of GMC(genetically modified crops) has developed on a large scale all over the world and the main Chinese GMC turned into industrialization is insect-resistant cotton, which has bought great benefit to the society, but the safety of the transgene arouse more and more attention.
The system of GMC identification has been established in China and now we report a insect-resistant cotton identifying system according to the transgene safety application of China at the molecular level. This is a basis reaserch of insect-resistant trangenetic cotton industrialization and our research may benefit to the molecular identification of GMC in china. The results are as below:
1.A modified method for extracting total DNA from cotton has been accquired. This method can eliminate the disturbance of polysaccharide and polyphenol which is very rich in cotton effectively and the total DNA obtained by this method showed to be more integral and pure. The ratio of OD260 and OD280 is about 1.8 and the yield is about 15ng DNA per milligram tissue.
2.We have established a PCR system which can rapidly identify Chinses insect-resistant cotton and America insect-resistant cotton. The optimal amount of the template is 50ng and the suitable condition is touch-down PCR:
preequilibrated the PCR system to the denaturation temperature 94℃ for 7min and then,
94℃ denaturation for 45sec, 63℃ annealing of primer for 45sec, 72℃primer extension for 1min, 2 cycles；
94℃ denaturation for 45sec, 61℃ annealing of primer for 45sec, 72℃primer extension for 1min, 2 cycles；
94℃ denaturation for 45sec, 59℃ annealing of primer for 45sec, 72℃primer extension for 1min, 2 cycles；
94℃ denaturation for 45sec, 57℃ annealing of primer for 45sec, 72℃primer extension for 1min, 2 cycles；
94℃ denaturation for 45sec, 55℃ annealing of primer for 45sec, 72℃primer extension for 1min, 2 cycles；
94℃ denaturation for 45sec, 52℃ annealing of primer for 45sec, 72℃primer extension for 1min, 2 cycles；
Followed by:Final extension:72℃ for 7min
3.the the number of GFM cry1A copy in insect-resistant cotton genome has been detection
by the method of Southern blotting and we suggest the optimal amount of genomic DNA for Southern is 30μg. The southern blotting show Bt toxin gene in 8 insect-resistant trangenetic cotton harbing single gene lines and 和5 insect-resistant trangenetic cotton harbing double gene lines is 1 to 4 copies respectively.
4.The detection of GFM cry1A gene and cpti show the gene flow has not accur between insect-resistant transgenic cotton and other plant in the regions of insect-resistant transgenic cotton.
5. we have identify the expression of GFM cry1A gene at the protein level by special test strip and the result show GFM cry1A gene express differently in 13 insect-resistant trangenetic cotton lines.
Postgraduate: Sun Xin Majo: Botany
Supervisor: Prof. Hu Baozhong Prof. Guo Sandui
引文
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