Genetic Engineering Disease Resistance (rice Blast, Stripe Disease) Transgenic Rice

Rice is one of the most important crops in the world. But the diseases, particularly rice blast disease (RBD) and rice stripe virus (RSV), are worldwide, devastating diseases which have extremely serious impact on the production of rice in the world. Cultivating new and high quality varieties of rice which could have the resistance to disease infection is one of the main research topics of plant breeders, and also is one of the major issues related to people's daily lives.Gene Rs-AFP1, which coding product belongs toγ-thionin protein, has been isolated and cloned from the germinating of radish seeds. As a kind of antimicrobial peptides, the former research showed that they could inhibit the growth of various fungi. Resveratrol belongs to phytoalexin, which could be secreted by plants when the plants undergo a harsh environment or being infected by the pathogens. As a main regulated and rate-limited enzyme of resveratrol biosynthesis pathway, resveratrol synthase has an important role in regulating the resveratrol synthesis and metabolism of plants.In this study, aγ-thionin protein gene Rs-AFP1 and a resveratrol synthase gene PNRSC have been isolated and cloned from the germination of radish seeds and cDNA library of immature seeds of peanut respectively. These two genes were first analized by bioinformatics software, and further integrated into the modified binary vector of pCAMBIA1300 using ubiquitin as promoter and herbicide resistance bar gene as a selectable marker. The mature embyros of rice were transformed by the recombinated vector through agrobacterium-mediated transformation. 13 transgenic Rs-AFP1 plants and 7 transgenic PNRSC plants were then harvested. After the preliminary screened by herbicide Basta and molecular identification,we're sure that the 1,4,6,7,10,13 strains of 13 transgenic Rs-AFP1 plants and the 1,2,3,4,5,6 strains of 7 transgenic PNRSC plants are positive plants.Then the positive transgenic plant's total protein of each gene were further verified by the identification of resistance to rice blast in vitro and the detection of resistance in the case of natural disease in the field and the identification of transgenic plants resistance to rice blast indoor. The preliminary results showed that the total protein of the positive transgenic plants with a certain degree of inhibition to Magnaporthe grisea, and in the field under natural infection, the wide-type rice were infected with rice blast, but the positive transgenic plants were not which under the same growth conditions, and the indoor identification of transgenic plants resistance to rice blast showed that all the positive transgenic Rs-AFP1 plants except the 10 line appeared disease resistance,and all the positive transgenic PNRSC plants except the 2 line appeared disease resistance. Thus, both the products of Rs-AFP1 and PNRSC have abilities to improve the resistance and tolerance to plant diseases in some extent. Both of this two blast resistance genes have a certain value in rice cultivation.The genome of RSV is composed of four independent single-stranded RNAs. According to molecular weight, those RNAs are classified into four groups, named RNA1, RNA2, RNA3 and RNA4. The modified plasmid pUC19 with two pairs of isocaudamners enzymes is used as RNAi vector. The single-stranded RNA of RSVs were linked into this RNAi vector by sense and antisense directions intervaled with an intron, and a hairpin structure could be formed. And the single-stranded RNA of RSV in vivo will be silenced and lose the pathogenicity once the virus infected plants as plants has been transformed by RNAi expression vector according to RNA interference principle. In this study, we totally designed 12 pairs of primers based on the RSV's four single-stranded RNAs. After RT-PCR and PCR amplification using RNA of RSV infected rice leaves as templates, finally 5 different cDNA fragments of RSV named OsRSV(1-5) have been cloned. The fragments then cloned into medial RNAi vector and finally subcloned into plant expression vector. The positive clones were identified and transformed into agrobacterium of LBA4404. The callus of mature embryos of rice"Shengdao 13", which is a main cultivars of Huanghuaihai rice production area, were used as acceptor material through agrobacterium-mediated transformation. We harvested 4 transgenic OsRSV1 fragment plants,6 transgenic OsRSV2 fragment plants,5 transgenic OsRSV3 fragment plants,7 transgenic OsRSV4 fragment plants and 11 transgenic OsRSV5 fragment plants. After the preliminary screened by herbicide Basta and molecular identification, the positive transgenic plants were identified , and then we got the detection of resistance in the case of natural disease in the field.The preliminary results showed that the trangenic plants have some abilities to improve the resistance and tolerance to RSV virus in some extent. And the insect inoculation and insect resistant abilities are be analyzed in the field and laboratory. The effective RSV fragments in insect resistance will be screened and selected for further rice breeding and new germplasm cultivation.In summary, the aim of our research is to resolve the major problems of rice diseases, especially rice blast disease and rice stripe virus, which were happened frequently in Huanghuaihai rice production area. By molecular cloning of some disease resistance-related genes, such as Rs-AFP1 and PNRSC, we got the transgenic rice plants by agrobacterium-mediated transformation. The preliminary screen by herbicide Baster combined with later molecular identification ultimately resulted in several positive transgenic lines for Rs-AFP1 and PNRSC genes seperately. And the preliminary observation and comparation of the incidence of RBD and RSV between transgenic plants with wild type ones showed that, the abilities of resistance and tolerance to disease were all improved in the field in some extent. And the disease inoculation and resistant abilities of both transgenic materials will all be analyzed in the field and laboratory further. Our research above will provid new materials for Rs-AFP1 and PNRSC gene function identification, and shed some light for new rice germplasm innovation of disease resistence in the future.