Primary Identification Of A New Resistant Gene To Bacterial Blight In Rice And Cloning And Expression Analysis Of Small Heat Shock Protein Gene

Rice is the main food crop in China.Bacterial blight(BB) which was caused by Xanthomonas oryzae pv.oryzae(Xoo) was one of the most serious plant diseases of rice (Oryzae sativa L.) production.Oryza meyeriana L.has high level of resistance to BB. Using somatic hybridization,special BB resistant gene in Oryza meyeriana L.was introduced into a cultivated rice variety 8411.SH5 and SH76 were the offspring of somatically hybridized rice.Using 11 BB races in Philippines and China,the BB resistant spectra of SH5, SH76 and other 16 varieties with different major BB resistant genes at three stages were evaluated.Genetic analysis was done through the reciprocal cross.The effects of BB on reactive oxygen species(ROS) metabolism of rice were conducted with SH76(highly resistant) and 8411(susceptible).The activities of disease resistant-related enzymes including superoxide dismutase(SOD),peroxidase(POD),catalase(CAT) and the content of H₂O₂ and malondialdehyde(MDA) in rice leaves were tested.An up-regulated gene sHSP 17.5 in somatically hybridized rice after Xoo inoculation was indentified using Affymetrix's gene chips.Reverse transcriptase polymerase chain reaction(RT-PCR) was used to analyze the temporal and spatial expression of sHSP 17.5. The full-length cDNA and DNA of sHSP 17.5 were cloned.The results are as follows:1.At the seedling stage,the resistant spectra of SH5 and SH76 were different from those of other formally identified resistance genes,but similar to those of IRBB5(xa5), IRBB7(Xa7) and Asominori(Xa17).At the tilling stage,the resistant spectra of SH5 and SH76 were similar to those of IRBB4(Xa4),IRBB5,IRBB7,Asominori and IRBB21(Xa21).At the booting stage,the resistant spectra of SH5 and SH76 were the same to that of IRBB21 and similar to those of IRBB5,IRBB7 and Asominori.2.From the results of the reciprocal cross,the resistance of SH5 and SH76 was controlled by a pair of dominant gene or linkage group,which could be a novel Xoo-resistant gene.3.The activity of superoxide dismutase(SOD),peroxidase(POD) and H₂O₂ content of resistant cultivar were higher than that in susceptible cultivar in 2 days after inoculation,and the peaks appeared at 2 days after inoculation.But the peaks of catalase (CAT) appeared at 3 days after inoculation.A negative relationship between net charge in malondialdehyde(MDA) content and resistance was found.These results showed that metabolism of ROS played an important role in the interactions of rice and bacterial blight.4.After inoculated with Xoo strain Zhe173,the expression patterns of gene sHSP 17.5 in leaves among the susceptible 8411 and resistant SH5,SH76 were different. sHSP 17.5 was up-regulated and stay in a high level in SH5 and SH76,but was down-regulated in 8411 at 24 h after inoculation.According to putative gene sequence in NCBI,specific primers were designed.A cDNA and DNA full-length sequence were obtained.The cDNA contains an ORF(501bp) encoding a complete protein.Genomic sequence of sHSP 17.5 did not have introns.Sequence analysis indicated that protein sHSP 17.5 contained four Alpha helix.Further study is needed to well understand its function.