Studies On Transgenic Rice Resistent To RSV Mediated By RNA-Interference

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. In recent years, greater losses were measured 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. RSV is transmitted only in the circulative/persistent manner by the small brown planthopper (Laodelphax striatellus Fallen) and three other delphacid species. As a typical member of genus Tenuivirus, RSV has filamentous particles which appear folded, branched and supercoiled in an electron microscope and it replicates in both plant and insect vectors. The molecular variability of CP genes of RSV was studied in our laboratory using 34 isolates collected from northern and eastern China. The identities of nucleotides of CP genes among these isolates were 96.8-100%, indicating that the sequence of cp gene was conservative for identified easily by siRNA.In the present studies, two RNAi vectors, namely p161=CP and p161=HCP were constructed. In these two vectors, the sense and antisense segements of the full length (969nts) or part (223 nts) of RSV CP gene were cloned into the pMCG161 plasmid respectively using the restrictive enzymes. There were an intron sequence between the sense and antisense segements, in order to product a intron-hairpin construct. The intron-hairpin construct involved 969 nts or 223 nts of the CP gene as the arms of the hairpin, between the sense and the antisense was a rice intron, derived from vector pMCG161. A fragment of the 7.5kb derived from vector p161=CP, which contained sense and antisense CP genes as well as intron-hairpin construct, was cloned into pCAMBIA1301 in order to adapt to the transformation system mediated by Agrobacterium tumefaciens. A new CP gene RNAi vector (named p1301=CP) was obtained based on pMCG161 and pCAMBIA1301, which could be used to transform plants mediated by A. tumefaciens. Vector p1301=HCP was also constructed. At the same time, a new plamid was obtained based on pMCG161 and pCAMBIA1301. This new plasmid could be used to construct the RNAi vector directly fitting for transformation system mediated by A. tumefaciens.RNAi vecors were transformed to callus induced by mature embryo of rice cv. Aichiasahi, Wanjing97 and Xiushui11 using A. tumefaciens EHA105. The targent gene was detected by PCR to confirm whether plants were positive. Finally eleven transgenic plants were identified. For the transgenic plants identification, the construct genes, such as GUS, NOS and 35S et al, were detected confirming whether those vector genes were lost or not in transgenic plants. The assay for viral resistance was performed by using viruliferous small brown planthoppers as inoculators. In order to ensure the pressure of inoculation, the ratio of viruliferous small brown planthoppers which showed 94.3 percent was firstly identified. After inoculating for 7 days with 10-12 planthoppers per plant, all the plants were then transplanted to the field or the greenhouse. The disease symptom of rice plants was investigated after 55 or 60 days. The appearance of disease symptom of transgenic plants was delayed by several days, as compared to nontransgecnic rice. Most of the transgenic plants only showed a lower or no rice stripe disease symptom. According to the criteria of resistance to RSV, Wanjing97 exhibited high susceptible to RSV infection, three lines of transgenic Wanjing 97(T2) showed moderately susceptible, seven lines showed moderately resistant, five lines showed high resistant. Based on the consecutive investigation of resistance to RSV of TO, T1 and T2 progeny of transgenic Wanjing97, the antiviral characters of T1 could be passed through to the T2 generation. Transgenic plants which showed invisible disease symptom were analysed that the content of RSV CP and its mRNA level were lower in those antiviral plants. In addition, the borders of T-DNA were analyzed on random transgenic samples. The results showed that foreign DNA inserted to rice genome with complexity of the situation, not in accordance with the T-DNA around the border. Furthermore, parts of the inserted vectors were broken and were integrated into rice genome with multi copies.