Producation Of Transgenic Rice Resistant To RSV And RBSDV By RNA Interference And Co-transformation

Rice is one of the most important cereal crops, contributing around40%of the national grainproduction and consumption in China. Rice stripe disease and rice black-streaked dwarf disease, causedby Rice stripe virus （RSV） and Rice black-streaked dwarf virus （RBSDV） respectively, occurs mainly ineastern Asia. They are both the most serious viral diseases in China, which are usually mix-infection,enormously dangerous and uncontrollable. With the deeper understanding of the RNAi mechanism, tocontrol virus diseases of plants mediated by RNA interference of efficiency and specificity have begunto receive attention and application. With the further research of genetically modified organism （GMO）and abundant GM crops putting into production, the safety assessment of GMOs has been improved.The co-transformation system of double T-DNA and two plasmids in two strains can be the practicablemethods to delete the selectable marker gene.In this study, pDT1301, a highly efficient plant expression vector was constructed basing on binaryvector pCAMBIA1301. By inserting a pair of the right and left border sequences of the T-DNA region,and changing the hpt II into the T-DNA2region, a Super-binary vector containing two pairs of T-DNAborder was established. In this vector, one of the T-DNA structural domain harbors the hpt II gene, andthe other harbors the Cauliflower mosaic virus35S promoter, Nos3′terminator and a multiple clone site.Theoretically, this vector can used to get transgenic plants without selectable marker gene.Taking advantage of a RNA interference vector pMCG161+/-D from our laboratory, whichcontains a partial nucleotide segment of RSV CP and RBSDV CP, we obtained RSV-RBSDV-HCP’shairpin by PCR. Then we connected the hairpin into the T-DNA1region of the super binary vectorpDT1301. The new Super-binary RNAi vector pDT1301-DR characterized in that the foreign gene andthe hygromycin resistance gene were located in different T-DNA regions, can be used to screeneddisease-resistant transgenic plants without hygromycin marker gene by selfing separation.Recombinant binary vector pDT1301-DR was introduced into the calli of a japonica rice varietyAichiasahi via Agrobacterium-mediated transfer. Filtered by hygromycin, we obtain227resistant callusand125T₀regeneration plants. Detected by PCR, there are31positive plants containing the target gene,the co-transformation rate is24.8%. Nineteen T₁transgenic lines were obtained after the self-pollination of the T₀transgenic lines with both the hpt II gene and target gene. Molecular detectionrevealed that14lines exhibited the antibiotic-free gene and were positive for the presence of the targetgenes. Southern blot analysis confirmed the integration of the exogenous gene into the rice genome withsingle copy, indeed prove foreign target-gene integrated into the genome of rice. Resistance analysis ofthe transgenic rice showed that the incidence of transgenic lines ranged from10.37%-41.00%, with anaverage incidence of29.50%. While the non-transgenic Aichiasahi and Huaidao5get serious disease,the incidence was up to100%.Taking advantage of a marker gene-free RNA interference vector pCMBIA1301+/-R from our lab, which contains the partial nucleotide segment of RSV CP, we attempted to improve the efficiency ofgenetic transformation in the test. The effects of both rice varieties and density ratio of two recombinantagrobacterium strains on the co-transformation efficiency were investigated. The results showed that therice variety Aichiasahi （9.07%） was more adaptive to co-transform system by RNAi vector than that ofthe cv. Wuyujing3（6.59%） and Huaidao5（0%）. Additionally, when the density of recombineagrobacterium E13R and E1301was3:1, the co-transformation efficiency was16.33%, higher than theothers, such as density ratio was2:1（13.56%）,1:1（9.09%） and1:2（5.97%）, respectively.A marker gene-free RNAi vector p1301-SP was constructed with disease-specific protein （SP）gene of RSV. Recombinant binary vectors p1301-SP were introduced into the calli of Aichiasahi viaAgrobacterium-mediated transfer. Filtered by hygromycin, we obtained192resistant callus underregeneration.