Isolation And Cloning The CDNA Fragment Of Rice Resistance-related Genes Against Magnaporthe Grisea Using DDRT-PCR

Rice blast,caused by the fungal pathogen Magnaporthe grisea, is one of the most serious diseases in rice (Oryza sativa L.) in the world ,which significantly reduced rice yield(Ou,1985).The use of resistant cultivars have been realized the most economical and effective way to control this disease. However, the effective lifespan of many cultivars is last only a few years, due to the breakdown of the resistance in the face of high pathogenic variability of the fungus (Kiyosawa, 1982). Moreover, with little understanding of the mechanism of disease resistance, It is very difficulty to cultivate wide and long-life resistance cultivars through traditional methods .Cloning rice resistance genes through genetic engineering and molecular biology technique, deeply understanding the interaction between pathogeny and host and further to analyze its characteristic of gene expression of rice is very importance to cultivate resistance blast rice. Inheritance of rice blast resistance has been extensively studied, up to date, at least 20 main effective genes and 10 QTLs have been mapped, with only two blast resistant genes (Pi-b and Pi-ta) have been cloned.Digu, an excellent rice blast resistant cultivar, was used in this research. The purpose of this research is to search for the difference expression genes before and after the treatment of Magnaporthe grisea(ZB\3) through DDRT-PCR, and further understand the mechanism of rice blast resistant.Rice seeding(3-4 leaf period) were inoculated by spraying with the pathogen at the conidia concentration of SxlOW"1 ,then extract rice leaf at differential periods. Using mRNA differential display technique to isolate differentially expressed cDNA fragments from rice leaf induced by inoculum of Magnaporthe grisea .87 differential cDNA fragments were obtained. After recovery and dot-blot reconfirm, 6 fragments were sequenced and named SSR-8,SSR-10,SSR-11,Z4,Z5 and Z7,respectively. Blastn and Blastx in GeneBank were compered with the former sequences.The main results are summarized as below:1. SSR-8 is highly homological (99%) with Oryza sativa genomic DNA, chromosome 4, BAC clone: H0302E05 and oj991113₃0 in the nucleotide sequence. Through BLASTX, ssr-8 ishomological with putative malate synthase (accession no: CAD79703) [Oryza saliva (indica cultivar-group)],the identities of amino acid is 78% .2. SSR-10 has 99% homology with Oryza saliva genomic DNA, chromosome 1 PAC clone: AP003291.4 and AP003294.3 in the nucleotide sequence. With BLASTX, there is not any significant similarity to be found, so it maybe a new gene fragment.3. SSR-11 is homology with Oryza sativa ssp. japonica cv. Nipponbare BAG genomic sequence OSJNBa0085I10,the identities is 97% ;and it is also highly homology with Oryza sativa (japonica cultivar-group) genomic DNA, chromosome 1, PAC clone:P0468B07 and AP003247.4.And through BLASTX , there is not any significant similarity to be found ,so it maybe a new gene fragment.4. Z4 is homology with Oryza sativa ssp. japonica cv. Nipponbare BAG genomic sequence OSJNBa0044D15, Identities = 95% .Z4 has 89% homology with Oryza sativa (indica cultivar-group) mRNA for RPRlh, complete cds and it's counterpart RPR1 in japonica cultivar-group .The BLASTX result also shows highly homology(75% identity) with RPRlh and RPR1. RPRlh and RPR1 have a conservative structure of NBS-LRR domain which is the characteristic of plant resistant genes. Furthermore, an ORF in the upward of z4 in the OSJNBa0044D15 is homology with a serial of plant resistant genes with NBS-LRR domain. All of this strongly suggest that z4 gene has the NBS-LRR structure and belongs to R gene family.5. Z5 is highly homology with Oryza sativa chromosome 12, BAG AL731891.the identities is 100%.The BLASTX result indicated no significant similarity in the GeneBank, so it maybe a new gene fragment.6. Z7 is highly homology with Oryza sativa (japonica cultivar-group) genomic DNA, chromosome 6, BAG clone:OSJNBa0004I20 and AP000616.1 in the nucleotide sequence;...