转基因抗虫棉花重组DNA在土壤中分布的实时定量PCR分析

根据转基因抗虫棉花35S启动子与Cry1A（c）基因以及35S启动子与nptⅡ基因之间的构建特异性序列分别设计引物和探针,建立荧光定量PCR检测方法,并对采集于3个生长时期（播种后40、50d和60d）的不同根区（根表、根际和非根际）土壤中35S-Cry1A和35S-nptⅡ片段进行定量分析。结果表明,所建立的荧光定量PCR方法最低能够检测到10个拷贝数的外源重组DNA片段,定量标准曲线相关系数均达到0.998以上,具有很好的重复性。实时定量PCR分析表明,同一生长时期不同根区土壤中35S-Cry1A和35S-nptⅡ片段拷贝数变化情况均为根表土〉根际土〉非根际土,即转基因抗虫棉花重组DNA片段主要分布于根表土壤中,其次为根际土壤和非根际土壤。在60d生长期内,土壤中35S-Cry1A和35S-nptⅡ片段的拷贝数随生长时期的推进均呈现上升趋势,其分布范围逐渐扩大。土壤中35S-nptⅡ片段拷贝数均高于同一生长时期相应根区中35S-Cry1A片段的拷贝数。转基因抗虫棉花对环境可能存在潜在影响。

Real Time PCR Assays for the Distribution of Recombinant DNA of a Transgenic Insect-resistant Cotton in Soil

During the growth process,genetically modified crops continuously release recombinant DNA to the soil environment.The uptake and integration of the recombinant DNA by soil microorganisms will alter the structure and function of the soil microbial community,and may cause long-term perturbation of the soil ecological system.Our objective is to establish quantitative PCR detection method to explore the distribution of the exogenous recombinant DNA in soil environment,to enrich the research contents of the environmental risks of transgenic insect-resistant cotton,and to provide important technical support for the scientific evaluation of ecological security of transgenic insect-resistant cotton.Primers and Taqman probes were designed according to the construct-specific sequences between 35S promoter and Cry1A（c） gene and between 35S promoter and nptⅡ gene respectively.The standard curves were prepared based on the linear relationship between the amount of input pGEMT-easy plasmid containing the construct-specific sequences（x）and cycle threshold（y）in 10-fold dilution mode.Three-room rhizobox method was used to collect the soil samples from different root zones of transgenic insect-resistant cotton.Total soil DNA was extracted by the PowerSoil DNA Isolation Kit.The quantity of exogenous recombinant DNA in soil samples was detected by the real time PCR method.The results showed that the established method could at least detect 10 copies of the recombinant DNA fragments,and the correlation coefficients were above 0.998 with good repeatability.All the copy number of 35S-Cry1A and 35S-nptⅡ fragments at the three growth stages showed the trend as follows：rhizoplanerhizospherenon-rhizosphere,which indicated that recombinant DNA fragments concentrated in the rhizoplane soil,followed by the rhizosphere soil and non-rhizosphere soil.During the 60 d growth period,the copy number of the 35S-Cry1A and 35S-nptⅡ fragments increased with the growing stages,and the distribution areas gradually expanded.The copy number of 35S-nptⅡ fragment was higher than 35S-Cry1A fragment in the same growth stage and in the corresponding root zone.Transgenic insectresistant cotton may have potential impact on the environment.