Cloning Of Pathogen-responsive Genes From Rice Induced By Bacterial Blight Pathogen, Xanthomonas Oryzae Pv. Oryzae And Construction Of Over-expression Vectors

Rice is an important crop worldwide. Bacterial blight (BB) caused by Xanthomonas oryzae pv.oryzae (Xoo) is one of the most serious bacterial diseases in rice. Cultivation of disease-resistant rice cultivars has been proved to be most economical, effective and environment-friendly strategies for control of the disease. Therefore, the isolation and cloning of disease-resistant genes becomes the foundation of rice resistant breeding.Wild rice is an important genetic resource, which contains various disease-resistance genes. O. meyerian (Oryza meyeriana Baill.) is highly resistant to rice bacterial blight disease. Based on the results of previous studies, O. meyeriana does not have disease-resistant gene Xal, Xa21, and other known disease-resistant gene. It possibly contains some new bacterial blight resistance genes and worthy of further study. However, the reproductive isolation and lacking of knowledge on the genetic background makes it impossible for cross-breeding and cloning of resistance genes by common methods. However, homology cloning provides a possible approach to clone the disease-related genes from the wild rice.When plants were infected, the pathogen was recognized by the disease-resistant genes, and a series of signal transduction processes started since the systemic acquired resistance (SAR) was induced in plants. In this process, a number of transcription factors and related genes were involved. Based on homology cloning, two homologous genes of the TGA family(OmTGA1,OmTGA2), a PR protein homologous gene(OmPR1) and xa5 homologous were cloned from O. meyeriana. The over-expression vectors of these genes were constructed and transferred to rice by Agrobacterium tumefaciens-mediated transformation. Transgenic plants were generated and verified by PCR analysis. The main relults of this study are as follows:1. Based on bioinformatics analysis, four genes'ORF were cloned from O.meyeriana by RT-PCR with degenerate primers. The genes were named as OmTGAl,OmTGA2, OmPRl and Omxa5, the size of these genes were 993bp,1410bp,486bp and 321bp, respectively.2. According to the sequences of ORFs that have been cloned, the full-length cDNA of the four genes were cloned with rapid amplification of cDNA ends (RACE) method. The sizes of these cDNAs were 1729bp, 1961bp,778bp and 842bp respectively, and the sequence analysis were also performed.3. Four over-expression vectors, containing each of these genes were constructed.4. These over-expression vectors were then transferred into rice (Nipponbare and Ishikari palemane) by Agrobacterrum tumefactens-mediated transformation, and the To generations of transgenic plants were verified by PCR analysis.