Breeding Of Transgenic Rice Resistance To Sheath Blight

Sheath blight was one of the three major diseases of rice. It caused great losses in rice production every year. The breeding progress for sheath blight resistance was very slow and so far no break-through had been made. The major reasons could be:(1) no rice varieties had been found to be completely immune to Rhizoctonia solani;(2) no major resistance QTLs had been identified to sheath blight;(3) lack of new resistance genes for transgenic rice breeding to sheath blight. In this study, we focused on the following two aspects:(1) bioinformatic analysis and functional characterization of the rice-derived polygalacturonase-inhibiting protein (OsPGIP) gene family that could be resistant to rice sheath blight;(2) transformation of8val genes that were essential for the biosynthetic pathway of validamycin A into rice. On one hand, we expected that we could identify important endogenous defense-responsive genes, which had potential applications in transgenic breeding against rice sheath blight. On the other hand, we aimed to try a new approach for rice sheath blight resistance transgenic breeding. The main results obtained in this study were as follows:1.2OsPGIP genes were newly identified in rice by using bioinformatic analysis, which all have the typical LRR domain, signal peptides directed to the membrane and conserved cysteine residues.2. An unrooted phylogenetic tree was generated by using the alignments of the PGIP amino acid sequences from different species. The results showed that all the PGIP from the Gramineae among monocot plants were in the same group, while the PGIP from monocot and dicot had a far relation. All these results might provide an evidence for the genetic evolution of PGIP from different species were conserved.3. The expression profiles of OsPGIP gene family (OsPGIP’3not included) in different tissues and organs throughout the entire life cycle of indica rice variety MH63were analyzed. The results showed that the expression patterns of these6genes were variable, which indicated that these6genes might play different roles in the development of rice.4. In order to confirm the results of the microarray database, we treated indica rice MH63with GA3, KT and NAA at trefoil stage. The expression profiles of these7genes were analyzed. The results showed that all7genes could respond to GA3, KT and NAA treatments. We also detected the responses of OsPGIP genes in japonica rice Zhonghua11to ABA, BR, GA3, IAA, JA, KT and SA treatments. The results showed that the responses of OsPGIP genes in japonica rice to different phytohormone treatments were complicated and diversified.5. In order to obtain the information about responses of the OsPGIP gene family in rice to Rhizoctonia solani, we inoculated rice japonica variety ZH11with Rhizoctonia solani at booting stage. The results showed that except of the decreased expression of OsPGIP6, all the other six genes were up-regulated after inoculation.6. To evaluate the different expression patterns and various responses to stresses of OsPGIP genes in japonica rice, putative cis-elements in the promoter regions were checked. The results showed that most OsPGIP promoters contained at least one cis-acting regulatory element associated with pathogen or phytohormone responses, which was in accordance with the different expression patterns of OsPGIP genes.7. We analyzed the subcellular localization of OsPGIP1,2,3and4by bombardment of the onion epidermal and transformation rice protoplasts by using PEG. The results showed that all four genes were localized on the cell membrane.8. We transformed OsPGIP1,2,3and4into rice and analyzed the transgenic plants by using southern blotting and northern blotting. The results showed that all four genes were integrated into the rice genome, and in some transgenic plants, the expression of each gene was significantly increased compared to the control plants. We selected highly-expressed transgenic plants with a single copy for follow-up studies.9. The resistance level of transgenic plants to Rhizoctonia solani was tested for two years in the field trial. Results showed that some of the OsPGIP1and4transgenic lines were more resistant to sheath blight compared to the negative transgenic control, the wild-type control and the susceptible variety control. This indicated that OsPGIPI and4might be useful for application in transgenic breeding against rice sheath blight.10. In addition, in order to build a novel validamycin A biosynthesis pathway in rice, eight necessary genes were transformed into the rice genome. Those eight genes, each driven by a rice derived promoter, were first constructed, and then two expression cassettes were put into a transformation vector, resulting four transformation vectors. All these vectors were transformed to rice with Agrobacterium-mediated rice transformation method individually to obtain transgenic plants. Transgenic plants were then analyzed by southern blotting and northern blotting. Results showed that all eight genes had been successfully integrated into the rice genome, and the expression levels of target genes in some transgenic lines were very high. We selected highly-expressed transgenic plants with a single copy as hybridization parents.11. We pyramided all eight genes by genetically crosses and tested the hybrids by real-time PCR. Results showed that all eight genes were abundantly expressed in some transgenic lines, however, we could not detect the existence of validamycin A using present available methods.