转基因番木瓜pUC57-Papaya质粒标准分子的构建与适用性研究  

Construction and Application of Plasmid Reference Molecule pUC57-Papaya for Genetic Modified Papaya

作　　者：潘志文[1] 陈伟庭[1] 姚涓[1] 王声斌[1] 姜大刚[1] PAN Zhi-wen;CHEN Wei-ting;YAO Juan;WANG Shengbin;JIANG Da-gang(College of Life Sciences,South China Agricultural University,Guangzhou,Guangdong 510642,China)

机构地区：[1]华南农业大学生命科学学院,广东广州510642

出　　处：《计量学报》2023年第3期422-429,共8页Acta Metrologica Sinica

摘　　要：构建了含番木瓜2个内标准基因、4个筛选靶标及3个转化体特异性片段的质粒分子pUC57-Papaya,进行了适用性验证实验。应用普通PCR和实时荧光定量PCR对其进行互换性评估;使用质粒分子对2个样品进行转基因含量测定。结果显示:质粒分子全部9个目的片段按照设计排列,序列完全一致;全部9个基因片段普通PCR和实时荧光PCR方法均能正确扩增得到预期结果,特异性、灵敏度与基因组DNA一致;全部9个基因构建标准曲线做定量测试时,与基因组DNA标准曲线斜率无显著差异,线性相关系数均大于0.99,其中内标准基因Papain、NOS终止子、YK1601与55-1事件特异性片段的截距无差异;利用质粒分子测量2个样品的Papain、NOS终止子、YK1601与55-1事件特异性片段拷贝数,计算转基因含量,结果与基因组DNA一致;测序结果显示各片段拷贝数比值为1,质粒DNA拷贝数浓度为(2.54±0.10)×10^(6)copies/μL。构建的标准质粒分子可代替基因组DNA用于定量检测,也可作为转基因番木瓜定性和定量检测用标准物质。In order to solve the problem of lack of reference materials of transgenic papaya in China,a A plasmid pUC57-Papaya including two papaya endogenous reference genes,four screening targets and three event-specific fragments was constructed.After sequencing verification,the applicability of the plasmid reference molecule was tested.The commutability was analyzed by ordinary PCR and real-time fluorescence quantitative PCR.Then the plasmid molecule was used to determine the transgenic contents of 2 samples.The sequencing result showed that all 9 target fragments of the plasmid molecule were arranged according to the design,and the sequence was completely consistent.The ordinary PCR and real-time fluorescence quantitative PCR results showed that the plasmid molecule could correctly amplify and obtain the expected results,which were consistent with genomic DNA.There was no significant difference with the slopes of standard curves constructed by the plasmid molecule and genomic DNA for quantitative tests,and the linear correlation coefficients were greater than 0.99.There was no difference in the intercept of Papain,NOS terminator,YK1601 and 55-1 event specific fragment.The copy numbers of Papain,NOS terminator,YK1601 and 55-1 event-specific fragments and the transgenic contents of two samples were measured by plasmid molecule,the results were consistent with genomic DNA.The sequencing result showed that the ratios of the copy number of each exogenous gene fragments to the copy number of the endogenous reference gene fragments were 1.The plasmid DNA copy number concentration was(2.54±0.10)×10^(6) copies/μL.The above results suggested that the constructed standard plasmid molecule could be used as a reference material for qualitative and quantitative detection of transgenic papaya.

关 键 词：计量学 转基因番木瓜 质粒标准分子 核酸标准物质 

分 类 号：TB99[一般工业技术—计量学]