基于质粒分子的转cry1Aa基因棉花定量PCR方法的建立  

A RT-PCR Methodology Based on a Constructed Plasmid Molecule for Detecting Transgenic cry1Aa Cotton

作　　者：苏长青[1] SU Changqing(Department of Life Science,Hengshui University,Hengshui,Hebei 053000,China)

机构地区：[1]衡水学院生命科学系,河北衡水053000

出　　处：《福建农业学报》2020年第6期569-575,共7页Fujian Journal of Agricultural Sciences

基　　金：衡水学院高层次人才科研启动基金项目(2018GC19)。

摘　　要：【目的】转cry1Aa基因棉花南农6号为我国未经批准商业化种植的棉花品种。为监测其非法种植,解决阳性标准品不容易获得的问题,构建适合转cry1Aa基因棉花南农6号品系特异性检测的质粒分子pMD-NN6,以该质粒分子作为标准物质,建立相应的实时荧光定量PCR方法。【方法】根据南农6号转化体特异序列和棉花内标准基因acp1设计引物,采用重叠PCR方法构建质粒分子;以质粒分子作为标准物质,南农6号转化体特异序列为靶标,建立了南农6号棉花特异性定量检测方法。【结果】构建的质粒全长为3148 bp,含有南农6号品系特异性序列和棉花acp1基因序列两个靶标片段的质粒分子,定量标准曲线斜率和扩增效率均符合要求,定量检测限为30拷贝。两个已知南农6号含量的混合样品(2.0%和0.5%)定量检测的准确度标准偏差均在±25%范围内,精确度相对标准差均≤25%。【结论】建立的PCR定量检测方法可以用于含有南农6号转基因棉花产品的品系特异性定量检测,所构建的标准质粒pMD-NN6可以代替其基因组DNA作为标准物质使用。【Objective】A real-time quantitative PCR method based on a constructed plasmid molecule was established to facilitate the detection of the illegal uses of the transgenic cry1Aa cotton(Gossypium hirsutum L.).【Method】Using overlapping PCR,a plasmid molecule,pMD-NN6,was constructed as the reference material(RM)to design primer pairs that targeted the sequences of NN6 and cotton endogenous acp1 gene.Accordingly,a specific quantitative PCR method was established to detect the presence of NN6 genomic DNA in cotton samples.【Result】The constructed plasmid molecule was 3148 bp in length consisting the sequences of NN6 and acp1.The slopes and the efficiencies of the standard curves for the established event-specific RT-PCR met the requirements for NN6 quantification.The limit of quantification was 30 copies.On the 2.0% and 0.5% mixed transgenic samples,the new methodology delivered an accuracy expressed as biases within ±25%,and a precision as relative standard deviation(RSD)≤25%.【Conclusion】The newly developed RT-PCR assay appeared to be adequate for the event-specific detection of NN6 products,and the plasmid molecule pMD-NN6 was considered applicable as an RM to substitute for NN6 genomic DNA.

关 键 词：抗虫棉 质粒分子 acp1基因 cry1Aa基因 品系特异性实时PCR 

分 类 号：S562[农业科学—作物学]