Resistance And Molecular Character Of Transgenic Rice Mediated By RNA Interference Against Rice Stripe Virus

Rice stripe disease is one of the most serious rice diseases and occurs mainly in temperate and subtropical regions. Losses of 20-30% have been observed commonly in japonica rice-grown regions in China. Greater losses have been measured since 2000 in Jiangsu, Shandong and Henan provinces of China due to climatic changes, changes in cultivation practices and loss of variety resistance. This disease is caused by Rice stripe virus (RSV), the typical member of genus Tenuivirus. Planting disease-resistant cultivars is the ideal way to control rice viral diseases. With the understanding of the RNAi mechanism, to control virus diseases of plants mediated by RNA interference of efficiency and specificity have begun to receive attention and application. Attempting to increase resistance to RSV via RNAi, a series of plant-transformation vectors, designed for generation of hairpin-RNA constructs were constructed, and then transformed successfully into a japonica rice variety Aichi Asahi by Agrobacterium-mediated co-transformation.The study included two sections as following. One is detection of molecular characters and resistant analysis to RSV of transgenic rice for screening of homozygous lines. The T1 and T2 transgenic rice seedlings were screened for Hygromycin resistance and then the resistance to RSV was identified by inoculation experiment. The disease symptom of rice plants was investigated and randomly sampled after 55 or 60 days. The target gene and alien elements in transgenic rice was detected by PCR to confirm whether plants were positive. Finally 38 transgenic plants were identified. According to the result of field resistance to RSV of transgenic rice, ten strains of each 13 lines showing immunity and high resistance (10 of lines T2 generation, 3 lines of T3 generation) were selected randomly and totally 130 strains had been detected with 129 strains positive. The T3 and T4 generation of 12 lines of transgenic rice were further detected. The result showed 127 plants as positive among the 130 transgenic rice strains. Most of the transgenic plants only showed a little or no stripe symptom. Finally 8 homozygous transgenic lines were obtained,and their hereditary stabilities were discussed as well. The other section was the study of inserted DNA structure of homozygous transgenic cp gene rice line 966-A-1-13 transformed by Agrobacterium-mediated method. The inserted foreign DNA located at position 32152046 of chromosome 1 (AP008207.1) of rice genome. It caused 15bps deletions at the target site, but did not cause any recombination in both 2.0kb-length regions of upstream and downstream direction to the insertion site when identified by PCR. Furthermore, the left and right border were both broken.