Production Of RSV-resistant Transgenic Rice By Twin T-DNA System And RNAi

The twin T-DNA system is a convenient and feasible method of deleting marker genes after filtration. the highly efficient RNAi plant expression vector pDTRSVSP was constructed and introduced into the calli (cv. Yujing 6 and double resistance transgenic rice SK) by Agrobacterium-mediated method, transformed tissue was selected in the presence of hpt. Regenerated plants were analyzed by PCR, southern blot and northern blot to obtain the selected antibiotic-free and highly RSV-resistant rice, which were used as the basis for RSV-resistant breeding.1. The highly efficient plant expression vector pCAMBIA1300DT was constructed basing on binary vector pCAMBIA1300. In this vector, one of the T-DNA structural domain harbors the hpt resistance gene, and the other harbors the Cauliflower mosaic virus 35S promoter and Polyubiqution gene of corn promoter, Nos 3′terminator, and a multiple clone site. Then, RSV cDNA fragment which was PCR amplified from 400bp RSV SP gene was cloned and inverted repeated inserted into the MCS respectively, and harboring intron gene of Rice PLD gene in the middle of them to construct the RNAi plant expression vector pDTRSVSP.2. Recombinant binary vectors pDTRSVSP were introduced into the calli (cv. Yujing 6 and double resistance transgenic rice SK) via Agrobacterium-mediated transfer. A total of 20 Yujing 6 and 18 SK transgenic lines were obtained. PCR analysis indicated that the average co-transformation frequency was 44.7%.3. T1 transgenic lines were obtained after the self- pollination of the T0 transgenic lines with both the hpt gene and target genes. Molecular detection revealed that 13.08% lines exhibited the antibiotic-free gene and were positive for the presence of the target genes. When the T1 generation self-pollinated, 6 Yujing 6 transgenic lines and 5 SK lines exhibiting the non-marker gene and harboring the target gene were regenerated through the hpt resistance and PCR tests.4. Resistance analysis of the transgenic rice showed that 3 Yujing 6 transgenic lines exhibited a highly enhanced resistance, whereas 2 SK transgenic lines were highly resistant against RSV, stem borers, and the herbicide PPT.5. Southern blot analysis confirmed the integration of the exogenous gene into the rice genome with low copies. Northern blot analysis revealed that all resistant transgenic plants exhibited a reduction in mRNA levels compared with susceptible plants; RSV specific short interference RNAs were present in the resistant transgenic plants. These results suggested that the virus resistance was reduced by RNAi.