The Xa5 Allele Transgenic Research Of Oryza Sativa And Oryza Meyeriana

Xanthomonas oryzae pv. oryzae (Xoo) causes bacterial blight of Oryzae sativa is the major constraints for rice. The use of resistant rice cultivars is the most enconomical and effective method to control this disease.Studies on plant resistance genes are one of the hostpots in the field of plant molecular biology. Until now, among many separeted resistant genes, only several recessive genes. The mechanisms of recessive genes can not be explained by those of dominant resistant genes, which have been universally accepted.38 bacterial blight resistance gene (27 have been localized and 5 recessive genes) in rice have been identified. However the xa5, in the general transcription factor IIA gamma subunit mapped on the short arm of the chromosome 5, was a typical recessive R gene in rice. The xa5 gene also interacted with the other dominant or recessive R genes with indicated specialty of disease resistant mechanism of xa5.The sequence of xa5 consists of a single condon substitution between the rice resistant varieties and susceptible. And the amino acid sites in the surface of the protein three-dimensional structure, and may be interacted with the the other proteins. Xa5 was the dominant allele of xa5.Os05g01710 (OsXa5) and Om05g01710 (OmXa5) were the homologous genes of the xa5 in the Oryza sativa and Oryza meyeriana. In the nucleic acid level have many nucleotide mutations, but at the amino acid level,OsXa5 was Val at position 39 to Glu, OmXaS was Val at position 39 to Glu and Ser at at position 81 to Glu. Colned OsXa5 and OmXa5 from rice to learn more about the gene function in plant disease resistance.1. The gene expression characteristics of OsXa5 and OMXa5Using the subcellular localization and BiFC conformed that OsXa5 and OmXa5 were nuclear-localized gene, and the OsXa5 and OmXa5 were not observed self-interaction phenomenon in the tobacco epiderman cells.To investigate the spatial expression patterns of OsXa5, SqRT-PCR analysis was performed.OsXa5 was highly expressed in leaves at tillering stage and exuberant stage in leaves conformed that the gene expressed in all organs and mainly expressed in the leaves. A variety of hormone treatments and environmental stresses to Oryza sativa L ssp. Japonica cv. Ishikari-shiroge (I-S),2 h and 24 h later tested the gene expression level by SqRT-PCR. The results showed that the gene expression levels have no significant differences between the two different time period. However, the amount of gene expression after 24 h under ET、PEG and high temperature stress were slightly changed compared to the 2 h, we speculated that OsXa5 may be involved in the response to the ET、PEG and the high temperature.2. The resistanceidentification and transcriptome of OsXa5 and PmXa5Over-expression of target genes has become an important method for the syudy of gene function in the mode crops such as rice. The OsXa5 and OmXa5 connected into the pCAMBIA1300UR-sGFP. With the PCR assay and chi-square test screening OsXa5-OE and OmXa5-OE. Compared with the I-S, differences in the seed germination of OsXa5-OE was not significant, the seed germianation rate of OmXa5-OE was lower. The leaves average number of OsXa5-OE was less and the OmXa5-OE was more, and the OsXa5-OE appeared aging leaves earlier than the OmXa5-OE. The tillers number of OsXa5-OE and OmXa5-OE was less than the I-S. The stem length, the length of the primary root and secondary roots of OsXa5-OE were significantly less than the I-S and 0mXa5-OE. The seed setting rate of I-S was significantly higher than the OsXa5-OE and OmXa5-OE, and the seed setting rate of OsXa5-OE was significantly higher than the OmXa5-OE.But the difference of the grain weight were not significant. Overall, over-express OsXa5 may leaded the dwarfish phenotype. We infered that the OsXa5 may influenced by the dwarf major gene and the GA pathway or BR pathways lead to the dwarfish phenotype. Over-express OmXa5 may lead the lower setting rate, may be because the OsXa5 from Oryza meyeriana, and reserved the good traits to some extent but the reasons has not yet to been known.The assay of OsXa5 in BB resistance by the leaf-clipping method was conducted at the maximum tillering stage, and were inoculated witOXoo (PXO99, PXO280 and PXO341). Disease was scored as the percent lesion area at 21 d post inoculation. The pathogenic differences between the three kinds, PXO99 was the strongest and the PXO341 was the weaskest. Over-expression of OsXa5 may slightly changed the resistance against bacterial blight. The OmXa5-OE enhanced the resistance to the pathogen and has the potential applications.Gene-Chip analysis reveals that 188 genes (probe sets) exhibited alterations in their RNA expression in OsXa5-OE plants. Among them,139 genes were up-regulated and 49 genes were down-regulated, contained 4 WRKY family genes,4 cytochrome P450 family gene,3 associated with the ethylene signaling way and 3 peroxidase enzymes genes and so on. The data of gene-chip was confirmed that the OsXa5 take many signaling pathways such as GA^ ABA and IAA and so on.3. The screening and verificate interacter factors of OsXa5 and OmXa5To further understand the specific interaction factor of OsXa5 and OmXa5, we have constructed the yeast two-hybrid cDNA library. And the library quality testing to meet the standards of the library screening. Respectively to the OsXa5 and OmXa5 as the yeast two-hybrid bait protein. OsXa5 and OmXa5 were constructed tested for self-activation in the yeast two-hybrid assay (the 3AT concentration was 25 mM) before them were used for library screens. An initial screening of 73 potential positive clones of OsXa5,59 potential positive clones of OmXa5. Verification by the sequencing and BiFC,4 colonies were interacted with OsXa5 in the tobacco epidermal cells, the similar to Histone H2A.2.1 (Os05g0461400), the similar to ubiquitin (Os09g0420800), the similar to Photosystem II (PS II) the oxygen-evolving complex protein 1 (Os01g0501800) and the catalase isozyme A (Os02g0115700).3 colonies were interacted with OmXa5 in the tobacco epidermal cells, the similar to Photosystem II 10 kDa polypeptide, the chloroplast precursor (Os07g0147500), the similar to light-harvesting complex I (Os06g0320500) and the another was the similar to glutathione-S-transferase Cla47 (Os10g0530500). From the body interaction lecel further confirmed OsXa5 involved in a variety of plants resilience signaling pathways.OmXa5 interacted with an effector in photosynthetic pathways, indicating that the OmXa5 may be related to the photosynthesis mediated signaling pathway, and participated in the process of the glutathione peroxidase defense ROS-mediated the plant anti-disease reactions.4% The prokaryotic expression and GST Pull-Down of OsXa5 and OmXa5Yeast two-hybrid and BiFC were in vivo to verify the interaction between factors, but in vitro the interaction is not yet known, and thus by building OsXa5 and OmXa5 fusion protein with GST tag. L-Arabinose and IPTG induced the expression of the fusion protein in the supernatant at low temperature and cruding the I-S crude protein, carried out the GST Pull-Down experiment. To identify differences in protein bands detected by SDS-PAGE, tapping recovered after the determination of the protein sequence. Trying to identify OsXa5 and OmXa5 in vitro interaction factor, through a comparative analysis to provide a basis for in-depth to explore xa5 and Xa5 machansim in plants, and can be provide the basis for the rice molecular breeding.