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利用根箱法对转基因抗虫棉花根部土壤进行分区采集,并采用新建立的土壤中转基因抗虫棉花重组DNA的半定量PCR检测方法对转基因抗虫棉花3个生长时期(播种后40、50和60d)不同根区土壤中内参磷酸果糖激酶(PFK)基因片段、35S-Cry1A构建特异性片段和35S-NPTII构建特异性片段进行分析,探索转基因抗虫棉花重组DNA在土壤中的分布特点。结果表明:播种后第40天、第50天的全部根表、根际及1个非根际土壤样品中检测到磷酸果糖激酶基因片段,第60天的全部土壤样品中检测到磷酸果糖激酶基因片段;第40天、第50天各有2个根表和1个根际土壤样品中检测到35S-Cry1A构建特异性片段,而非根际土壤样品中未检测到35S-Cry1A构建特异性片段,第60天的全部根表、根际及1个非根际土壤样品中检测到35S-Cry1A构建特异性片段,35S-Cry1A构建特异性片段相对量变化与磷酸果糖激酶基因片段基本一致;第40天、第50天和第60天全部根表土壤样品和第60天全部根际土壤样品中检测到35S-NPTII构建特异性片段,而在其他土壤样品中各有2个检测到35S-NPTII构建特异性片段,35S-NPTII构建特异性片段相对量变化与35S-Cry1A构建特异性片段基本一致;35S-Cry1A和35S-NPTII构建特异性片段与内参磷酸果糖激酶基因片段在土壤中的分布特点相似,主要分布在根表和根际土壤中,并随着棉花生长期的推进分布范围逐渐扩大。
The root-box method was used to collect the root soil of transgenic Bt cotton, and the semi-quantitative PCR method of transgenic cotton with recombinant Bt cotton was used to detect the growth of transgenic Bt cotton at three growth stages (40, 50 and 60d) were used to analyze the PFK gene fragment, 35S-Cry1A-specific fragment and 35S-NPTII-specific fragment in different rhizosphere soil so as to explore the distribution characteristics of transgenic insect-resistant cotton recombinant DNA in soil. The results showed that the phosphofructokinase gene fragment was detected in all rhizosphere and rhizosphere soil and one non-rhizosphere soil sample on the 40th day and the 50th day after sowing, and the phosphofructokinase gene was detected in all the soil samples on the 60th day The 35S-Cry1A-specific fragments were detected in two rootstocks and one rhizosphere soil on the 40th day and the 50th day, respectively, but not in the rhizosphere soil samples , 35S-Cry1A-specific fragment was detected in all rhizosphere and rhizosphere soil and one non-rhizosphere soil sample at the 60th day. The relative amount of 35S-Cry1A-specific fragment was basically the same as that of the phosphofructokinase gene fragment. 35S-NPTII-specific fragments were detected in all rhizosphere soil samples on the 40th day, the 50th day and the 60th day and in all the rhizosphere soil samples on the 60th day, while two of the 35S-NPTII were detected in other soil samples Construction of specific fragments, 35S-NPTII construction of specific fragments relative changes and 35S-Cry1A construction of specific fragments is basically the same; 35S-Cry1A and 35S-NPTII construction of specific fragments and the internal reference phosphofructokinase gene fragments in the soil distribution characteristics Similar, the main points In the root surface and rhizosphere soil, and gradually expand with the advance distribution of cotton growing season.