| RNA-mediated virus resistance is known as pathogen-derived resistance (PDR) and is one kind of posttranscriptional gene silencing (PTGS). It has been demonstrated that it is one of the most potent and efficient strategies in genetic engineering of virus resistance. Matrix attachment regions (MARs) are DNA sequences in eukaryotic chromosomes that bind specifically to nuclear matrix and have been proposed to organize chromatin into loop domains. Previous reports suggest that MARs may enhance transcription and can improve and stabilize transgene expression. Utilization of matrix attachment regions (MARs) in construction of gene expression vector provides an effective approach to enhance gene expression in transgenic plants. In this study, based on the tobacco species NC89 and the coat protein (CP) gene of potato virus Y (PVYN), different transgenic plants were generated with different plant expression vectors constructed with full-length untranslatable CP gene of PVYN or with 98bp inverted repeats(IR) with or without different MARs flanking the expression box. The resistance of these transgenic plants to PVYN was investigated. The main results and conclusions are as follows: 1. Effects of the different MARs on the resistance of transgenic plants induced by single-strand full-length untranslatable PVYN- CP gene (1) Vector pROKII and full-length untranslatable PVYN-CP were used to construct plant expression vector pRPVYCPN and other two plant expression vectors pRTM1CPNTM1 and pRTM2CPNTM2 which were flanked with different MARs. (2) Tobacco species NC89 was transformed with three plant expression vectors using leaf disk via Agrobacterium tumefaciens containing plasmid pRPVYCPN, pRTM1CPNTM1, and pRTM2CPNTM2, respectively. The transformed tissues were screening in the presence of 100mg/L Kanamycin. After tissue culture and screening, we got 72, 369 and 344 transgenic plants, respectively. The result of symptom observation and ELISA detection indicated that only 6 of 72 of transgenic plants transformed with pRPVYCPN showed high resistance to PVYN with the resistant ratio of 8.3% and that 58 of 369 in the pRTM1CPNTM1 lines and 52 of 344 in the pRTM2CPNTM2 lines showed higher resistance to PVYN with the resistant ratio of 15.7% and 15.1%. From the result of the resistant ratio, we can see that MARs can improve the resistance of transgenic plants in contrast with the control lines and there is no difference between the different MARs. Southern blot analysis and PCR of target segment in the genome of tobacco showed that all of the plants generated were the transgenic plants. Northern blot analysis showed that the level of the transcription from PVYN-CP was hardly detected in all the resistant plants, whereas the RNA in susceptible plants was accumulated to higher levels than that in the resistant plants, which indicated that the resistance of the resistant plants was RNA- mediated. The utilization of MARs in RNA-mediated resistance has not been reported so far. The result indicates that MARs can improve the resistance induced by single-strand full-length PVYN-CP, but there is no difference between different MARs. 2. Effects of the different MARs on the resistance of transgenic plants induced by IR. The 152bp and 98bp fragment from 3'end of PVYN-CP were used to construct inverted repeats. Two fragments were joined and cloned into the corresponding position of plasmid pROKII to generate the plant expression vector pRIR250. Two other plant expression vectors pRTM1IRTM1 and pRTM2IRTM2 were constructed based on the pRIR250 with different MARs. Tobacco species NC89 was transformed using leaf disk via Agrobacterium tumefaciens containing plasmid pRIR250, pRTM1IRTM1 and pRTM2IRTM2, respectively. The transformed tissues were also screening in the presence of 100mg/L Kanamycin. After tissue culture and screening, we got 159, 468 and 216 transgenic plants, respectively. Most of the transgenic plants transformed with pRIR250 showed high resistance after three times inoculation with PVYN. The result of symptom obser... |
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