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| Construction and Application of Plasmid Reference Molecule pUC57-Papaya for Genetic Modified Papaya |
| PAN Zhi-wen,CHEN Wei-ting,YAO Juan,WANG Sheng-bin,JIANG Da-gang |
| College of Life Sciences, South China Agricultural University, Guangzhou, Guangdong 510642, China |
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Abstract 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)×106copies/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.
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Received: 01 August 2022
Published: 08 March 2023
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2023, Vol. 44 
