利用根箱法解析转基因抗虫棉花重组DNA在土壤中的分布

Distribution features of transgenic insect-resistant cotton’s recombinant DNA in soil

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。

By using three-room rhizobox method, this paper studied the distribution features of the recombinant DNA of transgenic insect-resistant cotton in different zones of the cotton soil. A semi-quantitative PCR technology for detecting the recombinant DNA in soil was developed to investigate the distribution patterns of reference phosphofructokinase gene fragments and of 35S-Cry1A and 35S-NPTII construct-specific fragments in different soil zones at the three growth stages （40th, 50th, and 60th day after sowing） of the cotton. The phosphofructokinase gene fragments were detected in all rhizoplane, rhizosphere soil, and one non-rhizosphere soil at the 40th and 50th day after sowing, and in all soil samples at the 60th day. The 35S-Cry1A construct-specific fragments were detected in two rhizoplane and one rhizosphere soil at the 40th and 50th day, respectively, but none in non-rhizosphere soil, and detected in all rhizoplane, rhizosphere soil, and one non-rhizosphere soil at the 60th day. The relative amount of 35S-Cry1A construct-specific fragments had nearly the same variation trend as that of the phosphofructokinase gene fragments. The 35S-NPTII construct-specific fragments were detected in all rhizoplane at the 40th, 50th, and 60th day, and in all rhizosphere soil and two of the other soil samples at the 60th day. The variation trend of the relative amount of 35S-NPTII construct-specific fragments was basically the same as that of the 35S-Cry1A construct-specific fragments. Similar to the distribution of phosphofructokinase gene fragments, the 35S-Cry1A and 35S-NPTII construct-specific fragments mainly located in rhizoplane and rhizosphere soil, and the distribution areas of the recombinant DNA expanded gradually with the growth of the cotton.