| Rice stripe disease, with the pathogen Rice stripe virus (RSV), is one of the most widespread and severe virus diseases. In recent years, a quick spread of this disease had caused great losses in Huanghuai paddy fields due to changes in farming cultivation practices and technologies. Since 1999, the disease has broken out and become the major disease of this area. Infection of rice plants by RSV leads to the appearance of chlorotic stripes or mottling and necrotic streaks on leaves, with subsequent premature wilting which results in considerable decrease in grain yield. RSV is a representative of the genus Tenuivirus that is transmitted by a small brown planthopper, Laodelphax striatellus Fallen (Hemiptera, Delphacidae), in circulative-propagative and transovarial manners. RSV has filamentous particles which appear folded, branched and supercoiled in an electron microscope. A new powerful type of resistance, based upon the presence of RNA, known as RNA-mediated virus resistance (RMVR), was characterized by a high level of resistance that was not easily overcome by a high dosage of inoculums. In this study, we explore the molecular mechanism of RSV severer pathogenicity, and plant strong resistant and stably inherited T2 transgenic rice. The significant result can help increase the rice harvest and keep the sustainable development of rice product. The main results and conclusions presented in this thesis are as follows:(1) The analysis of molecular variability of RSV isolate (RSV-SD-JN2) in Jining ShandongTraditonal biology assay found that there is chemical composition and pathogenicity difference among different isolates of RSV. The difference expressed on the nucleotide level in recent years'study. According to the sequence analysis of RNA3, RNA4, or part encoding gene of some isolates, RSV gene sequence,5'or 3'untranslated regions are very conserved, the main variability occur in the intergenic region(IR). However, there is no systematic analyse of genetic variation among different virus population.A highly pathogenic Rice stripe virus was isolated from susceptible rice in Jining, Shandong Province, China, and was designated as RSV-SD-JN2. In order to explore the reason of RSV molecular variation and severer pathogenicity on the molecular level, we cloned the RNA3 and RNA4 cDNA fragments of RSV-SD-JN2 by reverse transcriptase-polymerase chain reaction (RT-PCR) and sequenced them. The results showed that the length of RNA3 and RNA4 of SD-JN2 were 2487bp and 2157bp respectively; the NS3 gene, IR and CP gene in RNA3 were 636bp,725bp and 969bp, respectively; the SP gene, and IR and NSvc4 in RNA4 was 537bp,654bp,861bp, respectively.Compared with the complete or part sequences of the RSV isolates from Panjin (PJ) in Liaoning province, Shuangqiao (SQ) in Beijing, Hongze (HZ) in Jiangsu, Kunming (KM) in yunan provice, Jining (SD-JN1) in Shandong, Jiangyin (JY) in Jiangsu province, ZHEJING in Zhejiang province that had been reported previously with different period, different area, different pathogenicity, we found that the most highly conserved regions located in 5'and 3' untranslated regions, which had only few base difference; the highly consensus of encoding regions also revealed more than 93% identity at the nucleotide sequence level, and more than 97% identity at the amino acid level, and most bases mutation was nonsense mutation; however, the most variable regions located in the intergenic region(IR). The variability of IR leaded to enhancement of RNA secondary structures-hairpin structures stability, which was the important reason of severer pathogenicity. Moreover, there is possible different genetic relationship among internal RNA3 different sequences of SD-JN2 isolate; the high homology of NS3 gene and CP gene compared with HZ isolate, is 98.9% and 99.1% respectively, but IR is 86.3%. It suggests that exchanged recombinant of different subgroup isolate cause the genome relevant fragment cause the result.(2) Production of transgenic rice new germplasm against two isolations of RSVCultivating a resistant breed is one of the most economical and efficient methods in preventing rice virus disease. However, a conventional method of breeding resistant varieties is a long-term and low-efficiency process. With the development of genetic engineering, transgenic technology has become a fast and efficient method of developing virus resistant varieties. RMVR, which has the advantage of high resistance (almost immunity), resistance durability, and high biosafety, has become a more tangible strategy to develop virus resistant plants. Although RMVR has a narrow resistance spectrum, transgenic plants can be produced by cloning effective nucleic acid fragments of different genes of the same virus, constructing plant expression vectors, and inserting into plants to extend resistance against different isolates of the same virus, apart from enhancing and stabilizing its genetic resistance. In this study, we generated stable, RSV-resistant, transgenic lines. The success in producing new germplasms of highly resistant transgenic rice plant will lay the theory foundation for breeding RSV-resistant rice to prevent resistance loss due to virus mutation.Expression vector pCAMBIA1300UNB was constructed with inserting Ubiquitin promoter and Nos-3'terminator, Changing hygromycin B resistance gene with Bialaphos resistance gene (Bar) based on vector pCAMBIA1300. Following RNA interference (RNAi) theory, we constructed three RNAi binary vectors based on coat protein(CP), special-disease protein(SP) and chimeric CP/SP gene sequence. Transgenic lines of rice CV. Yujing6 were generated through Agrobacterium-mediated transformation. We got T0 transgenic plants 23, 24 and 17, respectively.The self-fertilized seeds from T0 generation transgenic plants of transformed p1300CP/SP, p1300CP, and p1300SP vectors were planted to obtain T1 generation lines. In every transgenic line,100 positive plants were selected and inoculated with two RSV isolates SD-JN2 and JS-HA using viruliferous vector insects. Wild types (cv. Yujing 6) were used as control and then monitored daily for the appearance of symptoms. After four weeks of inoculation, lines CS1, CS3, CS6, CS7, CS10, CS13, CS15, CS16, and CS19 of p1300CP/SP transgenic plants; lines CP1, CP3, CP4, CP7, CP9, and CP12 of p1300CP transgenic plants; and lines SP2, SP5, SP6, SP7, and SP13 of p1300SP transgenic plants exhibited resistant phenotype, which had a susceptible score below 6%, far lower than the susceptible ratio of wild types. The susceptible ratios of other lines of the three vectors were all above 12%, exhibiting moderately resistant lines or susceptible lines. In these resistance assays chimeric CP/SP RNAi lines showed stranger resistance against two isolates than CP or SP single RNAi lines. The ratios of resistant transgenic lines were 39.13%,25.00%, and 29.41% in p1300CP/SP, p1300CP, and p1300SP, respectively. The result verifies that the ratio of T1 transgenic resistant lines containing CP/SP chimeric gene is higher than that contained the single CP or SP gene; furthermore, resistant lines containing CP/SP chimeric gene have the same high resistance to two RSV isolates.Stable integration and expression of RNAi transgenes were confirmed by Southern and Northern blot analysis of independent transgenic lines. In the resistant transgenic lines, relative to the susceptible transgenic plant, lower levels of transgene transcripts and specific short interference RNAs were observed, which showed that virus resistance was increased by RNAi.we selected T2 self-fertilized progeny of resistant transgenic plants which were Southern blotted proved have one copy from lines CS1, CS6, CP1, CP3, CP9, SP5, and SP7 to study the segregation pattern and stable inheritance of RNA-mediated multiple virus resistance. In the T2 generation, some resistant transgenic plants were stably homogenous. The resistance of transgenic plants would not separate at all, he results showed that transgene and RNA-mediated virus resistance can be stably inherited until T2.
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