Rapid And Accurate DNA-Based Detection Method For RNA Interference Transgenic Plants

RNAi technology is increasingly widely used in transgenic plants, including those allowed commercial production. But the rapid and accurate detection methods for RNAi transgenic plants are relatively lag behind those for common transgenic plants. The purpose of this study is to establish a rapid and accurate detection method for RNAi transgenic plants based on DNA technology, which can be used for effective detection of RNAi transgenic plants, and can make up for weaknesses in the current GM detection technology. Our detection system was established based primarily on preparation of probes, multiplex PCR, whole genome amplification, gene chip technology and gel electrophoresis after degeneration and renaturation of the production of PCR.37 GM elements commonly used in RNAi transgenic events were selected for analysis after consulting a large number of literatures for preparation of probes. These elements included 14 promoters, 4 terminators, 5 selectable marker genes, 2 reporter genes and 12 introns. Primers were designed for making probes according to conservative sequences in these GM elements. We also set up positive, negative and blank control as quality control for array hybridization. 18S rDNA widespread in eukaryotes was used as a positive control, while a gene expressed in the liver of boar as negative control and nucleic acid free spotting solution as blank control. Probes were obtained by PCR. They were loaded onto the chips after purification and quantitation.We collected eight plant samples, including four RNAi transgenic plants, two normal transgenic plants, one transgenic rice carrying empty vector and one wide-type rice. Genomic DNAs were extracted from these plant samples, amplified and analysed by hybridization to the microarrays made above. In one of the amplification methods, the GM elements were amplified specifically by using multiplex PCR. In the other one, the whole genome DNAs were amplified and fragmented before applying to the microarray.We optimized the system for multiplex PCR and whole genome fragmentation, and obtained samples suitable for array hybridization. We carried out array hybridization for multiplex PCR samples of OsBTF3, OsCtBPA, R1 and pTCK303, and for genomic sample of OsCtBPA. The hybridization results indicated that when the multiplex PCR samples were used for hybridization, the signals for the GM elements were relatively strong, when the whole genomic DNAs were used for hybridization, the signals for positive control, genomic elements of rice and GM elements were detected. The microarray method was successful, and the results could be used for the next step. But there were some false positives, and the microarray method remained to be improved.Gel electrophoresis after degeneration and renaturation was carried out by using RNAi transgenic plant OsCtBPA constructed by our lab. We designed primers for the amplification of RNAi constructs according to the promoters and terminators detected by array hybridization. Results indicated that the amplified RNAi constructs had shifed on gel after degeneration and renaturation, while the negative control with empty vector had no changes. Therefore, this method could be used to detect RNAi specific transgenic plants. More samples will be tested by this method in the future.According to optimization of the systems for preparation of probes and samples, making chips, array hybridization and gel electrophoresis after degeneration and renaturation, we established a preliminary detection method based on plant DNA for RNAi transgenic plants which took vital significance in the regulation of RNAi transgenic plants.