CDNA Cloning And Expression Of Rice OsFd And OsFNR Induced By Magnaporthe Grisea

The functional identification and expressing characterization of plant genes that are induced during interaction with pathogen will be a great help to reveal the mechanism of plant-pathogen interaction; Isolation of these genes may provide a good choice for engineering plant disease resistance. In this study, two differential displayed fragments, 21A03 and 21C01, were isolated from rice leaves (cv. Aichiasahi) infected by an incompatible race 131 of Magnaporthe grisea and used as probes to screen the cDNA library, which was constructed using mRNA from the p131 pathogen infected leaves. By this screening, two kinds of full length cDNA clones were obtained, of which, one is a Fd (ferredoxin) gene named as OsFd and the other is FNR (ferredoxin NADP+ oxidoreductase) gene named as OsFNR. The cDNA sequence of OsFd is 848bp and contains one intact open reading frame. The cDNA sequence shares 83% identity with that of maize FdIII from maize. It encodes a protein of 153 amino acid residues. The deduced amino acid sequence shares 81% identity with that of maize FdIII. OsFd transcript in rice leaves can be induced by compatible and incompatible blast fungus infection and SA. Genomic Rice genome contained one copy sequence homologous to OsFd gene. The size of nucleotide sequence of OsFNR is 1495bp, contained one open reading frame encoding 378 amino acid residues. Sequence analysis indicated that the cDNA sequence of OsFNR is identical to that of FNR from rice embryo. Northern blotting showed that OsFNR transcript in rice leaves can be induced by incompatible blast fungus infection and SA, suggesting that the OsFNR induction be involved in the defense of rice to M. grisea. Homologous analysis to Rice Genome Database suggests that OsFNR exists as one copy in rice genome. In order to further analyze the biochemical characteristics of OsFd and OsFNR the prokaryotic expressing recombination vectors pGEX-4T-3/OsFd and pGEX-4T-3/OsFNR were constructed and transformed into BL21 (DE3), respectively. The optimum expressing conditions for the both were approached, SDS-PAGE analysis showed that the products of recombinants OsFd and OsFNR were both specific. To clarify if OsFd and OsFNR are required for resistance in rice to blast, two types of rice expressing vectors (sense and hairpin) of OsFd and OsFNR were constructed and cloned into binary pCAMBIA 1301, and then transformed into rice by Agrobacterium tumefaciens-mediated transformation (cv. Aichiasahi), respectively. T0 transgenic rice lines of the four rice expressing vectors were obtained. There are 83, 110 ,43, 121 lines in transformation of sense and hairpin expressing vectors of OsFd and OsFNR , respectively. In conclusion, OsFd and OsFNR participate in the process of rice resistance to blast. On the other hand, we cloned the four genes from the glyoxylate cycle in rice. They are ICL( isocitrate lyase), CS(citrate synthase),MDH(malate dehydrogenase), MS(malate synthase) genes and they are named as OsICL,. OsCS ,OsMDH and OsMS, respectively. OsICL transcript in rice leaves can be induced by comapatible blast fungi infection.