| Establishment of Oryza sativa Specific ESTs Database Induced by Magnaporthe grisea and Identification of Resistance-related GenesPh. D. Candidate: Zhuang Xiaofeng (Plant pathology and Molecular biology) Directed by Prof. Li Debao1 In this study, a cDNA library from rice leaves induced by Magnaporthe griseawas sequenced randomly in a large scale. 15 396 ESTs were obtained, of which 14 975 have been released in dbEST (http://www. ncbi. nlm. nih. gov/dbEST). 5633 unique genes (42. 3 %) were identified through redundant analysis within 13 316 ESTs containing Poly (A) tail, among which 4503 genes were low redundancy (single or twice ) , 1074 genes were medium redundancy (3-19), and 56 genes were high redundancy (more than 20).The database of specific ESTs expressed in rice leaves induced by M. grisea was constructed for the first time by searching against Oryza sativa database in NCBI. In this database, 642 (11.4 %) were function annotated, 3628 unique genes (64.4%) were unknown, and other 1560 genes (27. 7%) were located in different chromosomes.It was found that 66 proteins were associated with plant resistance. Proteins were related to phenylpropanoid biosynthesis, oxygen metabolism, redox reaction, or belonged to pathogenesis-related protein, and some were resistant to stresses such as high temperature, chilling, light and wounding, etc. Therefore, it was concluded that rice defense response to disease was an complicated gene network, and closely related to biotic stress and abiotic stress.2 Interestingly, while compared to rice blast fungus genomic database and ricegenomic database, 16 genes originating from M. grisea were found from 5633 unique genes and picked out for further analysis. Bioinformatics analysis suggested that these gene were imported to pathogenesis-related development. Some of these genes, such as Cyclophilin (MgrOOS) and P-tpye ATPase(MgrOll), were involved in appressori urn formation. Galactose oxidase (Mgr007) and beta-glucosidase (Mgr016) were associated with recognizing host and decomposing host cell wall. C-4 methyl sterol oxidase(Mgr005) and delta(24)-sterol Omethyltransferase(Mgr009) were involved in the synthesis of ergosterol which is essential for pathogenicity of M. grisea. Plasma membrane sodium response 2(Mgr002) and serine/threonine kinase (Mgr015) may be involved in signal transduction during the infection. Ribosomalprotein L12 (Mgr014) and elongation factor 2(Mgr010) were important to protein synthesis. Coatomer (Mgr006) was attached to the trafficking of proteins and secretion within eukaryotic cells. CipB protein (MgrOOS) was involved in fatty acid biosynthesis. All the above genes may be involved in host-pathogen interaction in rice tissue.3 A cDNA array containing 1106 unique genes from 3 cDNA libraries was preparedto analyze the expression patterns of rice near iso-genic lines H7R (resistant) and H7S (susceptible). The results showed that 170 genes changed remarkably in 980 valid hybridization data, in which 106 genes were up-regulated and 64 were down-regulated after the rice samples induced by M. grisea for 8 h.In order to clone the new genes related to plant resistance to pathogen, ten genes up-regulated in cDNA array were analyzed further by bioinformatics. Except for caffeoyl-CoA 3-Q-methyltransferase (CCoAOMT) and cinnamyl alcohol dehydrogenase CAD), which were essential to lignin formation during lignif ication, all others may be participated in rice defense to disease. One clone CjOOleOQ) containing POZ/BTB domain was screened out for electronic cloning. By cloning in silico, a full-length cDNA sequence (1974bp) named OsBTByas cloned. A DNA sequence about 5174 bp was found through BLASTn to rice genome sequence. Northern hybridization confirmed that OsBTB was expressed at the early stages in response to pathogen infection.RNA array containing 755 rice samples was prepared to detect the expressions of OsBTB and 9 resistance-related genes (PAL, P-1, 3-glucanasc, polyubiquitin, SAM, SOD, phospholipase C, MARK, Pib, glycine-... |
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