Primary Identification Of A New Resistance Gene To Bacterial Blight From Rice And Cloning And Expression Analysis Of A Candidate Disease Resistance Gene

Bacterial blight(BB)caused by Xanthomonas oryzae pv.oryzae(Xoo) was one of the most destructive diseases of rice(Oryzae sativa L.).Oryza meyeriana L.shows high resistance or immunity to bacterial blight.Novel BB resistance gene(s)for rice was/were introduced into a cultivated japonica rice variety Oryza sativa L.cv.8411,via somatic hybridization using the wild Oryzae meyeriana as the donor of the resistance gene(s).SH5 and SH76 were the progenies of somatically hybridized plants which overcame the problem of fertile hybrids.The BB full-life stage resistance spectra of SH5,SH76 and other 16 reference lines carrying different major BB resistance genes were evaluated and compared using 11 BB races collected from the Philippines and China.According to the spectra,certain molecular markers were used to further identify the resistance gene(s)in the new germplasms SH5 and SH76.In the previous work of our lab,an up-regulated protein XooIARP in SH5 leaves after Xoo inoculation was identified by high resolution 2-dimensional electrophoresis(2-DE).Reverse transcriptase polymerase chain reaction(RT-PCR)was used to analyze the temporal and spatial expression of XooIARP.The full-length cDNA of XooIARP and the corresponding genomic sequence were cloned.The results are summarized as follows:1.The resistance spectra of SH5 and SH76 at the seedling stage were different from those of other identified resistance genes,but similar to those of IRBB5(xa5)and IRBB7(Xa7).At the tilling stage,SH5 and SH76 had the exact same resistance spectra as IRBB21(Xa21)did,and showed the similar ones to those of IRBB4(Xa4),IRBB5,IRBBT,Asominori(Xa17) and Minghui63(Xa25,Xa26).While at the booting stage,the resistance spectra of SH5 and SH76 were the same as that of IRBB21 and similar to those of IRBB5 and IRBB7.All in all,the spectra of two new germplasms were similar to those of IRBB5,IRBB7 and IRBB21,molecular markers were used to further detect whether there were xa5,Xa7 or Xa21 in SH5 and SH76.2.The sequence of marker 2F₁R was in one intron of xa5,and it was a functional marker.M5 was an InDel marker which was closely linked to Xa7.The presence of Xa21 gene could be detected by the STS marker pTA248 which was located within 1cM of Xa21.pTA248 could detect a band of 1 kb in all the resistance lines containing Xa21.The sequence of marker XA21 was also in Xa21.The result showed that there was no xa5, Xa7 and Xa21 in SH5 and SH76.3.The result of RT-PCR showed that the expression patterns of gene XooIARP in leaves among the resistant SH5,SH76 and susceptible 8411 were different after inoculation with Xoo strain Zhe173.XooIARP was up-regulated in two new germplasms with the same pattern as the translation level(previous work in our lab),while was down-regulated in 8411. According to the putative gene sequence submitted to NCBI,primers were designed.A 1302bp cDNA full-length sequence and a 2856bp genomic sequence were obtained.The cDNA contains an ORF(25—1050bp) encoding a complete protein.Genomic sequence of XooIARP comprised 11 exons with 10 introns conservatively inserted in the coding regions. Sequence analysis indicated that protein XooIARP contained two repeats of the N-terminal domain of Glutathione S-transferase(GST).Further study is needed to determine whether it functions as a GST.