| Rice bacterial blight, caused by Xanthomonas oryzae pv. oryzae (Xoo), is one of the serious bacterial diseases, has seriously affected the quality and yield of rice. Cloning resistance genes and developing resistance varieties is an economical and effective way to control the disease.We cloned 22 rice bacterial blight resistance-related genes. Four disease resistance-related genes were selected by using yeast two-hybrid; the other eighteen disease resistance-related genes were selected after inoculated Xoo to Y73. Xa5 is an important pivot of rice disease-related signaling pathways; xa5 is a known resistance gene, and the resistance of xa5 is originated from the different amino acid compare with Xa5; OmXa5 is the allele of Xa5, and the amino acid sequence of OmXa5 is different from Xa5 and xa5. In order to study the functions of these 22 genes, and the interactional relationship between these 22 genes and the rice bacterial blight resistance-related key genes Xa5, xa5, OmXa5, a series of experiments were taken.After 22 interactional candidate genes respectively interacted with Xa5, xa5, OmXaS by using BiFC (Bimolecular fluorescence complementation), we found:Xa5 interacted with catalase isozyme (Os02g0115700) and histone-like protein (Os05g0461400) mainly located in the nucleus, Xa5 interacted with ubiquitin-like protein (Os09g0420800) located in both nucleus and cytoplasmic membrane, and no interaction signals were observed when Xa5 interacted with other 19 candidate genes; The interactional pattern of OmXa5 was identical with Xa5; xa5 interacted with catalase isozyme (Os02g0115700) and histone-like protein (Os05g0461400) mainly located in the nucleus, and no interaction signals were observed when xa5 interacted with ubiquitin-like protein (Os09g0420800) and other 19 candidate genes, showing the interactional pattern of xa5 was different from Xa5. Thus, we speculated that the catalase isozyme (Os02g0115700), histone-like protein (Os05g0461400) and ubiquitin-like protein (Os09g0420800) are the important regulatory factors of rice disease resistance-related pathways. The functions of OmXa5 are similar with Xa5, the mutated amino acid may not affect the functions of OmXa5.The subcellular localization of 22 interactional candidate genes were examined to provide important information for the study of gene functions. Five genes combined with sGFP were found mainly located in the cell nucleus, showing the typical characteristics of transcription factor; Five genes combined with sGFP were found mainly located in the plasma membrane, indicating their potential functions in transport control on cell membrane; Six genes were simultaneously located in both cell nucleus and the plasma membrane and there were no apparent subcellular localization signals for the remaining 6 genes combined with sGFP.The rapid and flexible cloning method, Gateway recombination, was used in interactional identification and subcellular localization. But the low linearization efficiency and ligation efficiency of entry vector constructing had decreased the experimental velocity. Then the Gateway entry vector construction method was improved by using Bciâ…¥ to linearize the entry vectors. The linearization efficiency of Bciâ…¥ was significantly better than Xcml and there was also an about 40% increase in positive colonies in the testing experiment. The method was highly efficient for PCR products up to 2000 bp long (approximately 50% colonies containing insertions of the expected orientation). And can still be used, although with less efficiency, for fragments of up to 4700 bp. This improved entry vector construction method is stable and efficient in characteristics, and can make the Gateway entry vector constructing more efficient in the future. |
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