The Preliminary Analysis Of Rice Material Through RNAi Technology Against Rice Black Streaked Dwarf Virus

Rice black streaked dwarf disease is caused by the rice black streaked dwarf virus (RBSDV) and leads to serious damage against the food security. In this thesis, target genes from RBSDV and its media, small brown planthopper(Laodelphax striatellus Fallen) were isolated and used for making RNA interference (RNAi) vectors, which were respectively transformed into the rice cultivar wulingjingl to generate transgenic plants. We expect to obtain new transgenic lines displaying high resistance against RBSDV or the planthopper, and explore their anti-RBSDV mechanisms. The main results are as follows:1.5 RNAi vectors for RBSDV S4, S8 and S9 were constructed with an effective and efficient way "two-step method", and were respectively transformed into the susceptible rice cultivar wulingjingl, mediated by Agrobacterium tumefaciens EHA105.10 transgenic plants containing S9 RNAi fragment and 13 transgenic plants containing S8 RNAi fragment were generated. So far, we have not yet obtained transgenic plants with S4 RNAi fragment. T2 transgenic plants with S8 RNAi fragment were used to assess their resistance against RBSDV by viruliferous planthopper, and the results showed that, compared with nontransgenic plants, these transgenic plants displayed enhanced resistance to the virus.2. We spliced S9 with S4 or S8 to form a larger RNAi fragment S4&S9 (1 chimera) or S8&S9 (2 chimeras) by overlapping PCR, and 66 transgenic plants with RNAi fragment S8&S9 and 8 transgenic plants with RNAi fragment S4&S9 were respectively generated.3. We further maked 14 RNAi constructs respective for seven key genes from the small brown planthopper, including receptor for activated protein kinase C gene (RACK),60S ribosomal protein L5 gene (RPL5), glyceraldehyde-3-phosphate dehydrogenase gene (GAPDH3),60S ribosomal protein L7a gene (RPL7a),60S ribosomal protein L8 gene (RPL8), NADH dehydrogenase subunit 4 gene and NADH dehydrogenase subunit 5 gene (ND4 and ND5), and NADH dehydrogenase subunit 2 gene (ND2). These constrcuts were respectively transformed into wulingjing1, and most of them generated positive T1 transgenic plants.4. We established effective RBSDV inoculation system in the greenhouse with viruliferous planthopper, which can transmit the virus from isolated rice leaves or living rice seedlings into target seedlings.