Isolation And Functional Characterization The Pathogen-inducible And Tissue-specific Expression Promoters

The control of gene's expression is important mechanism for plants and animals. It is the basis of physiology, metabolism, development and reproduction of organisms. It is the hotspot of molecular biology on what is the expression controlling mechanism of genes. Promoter is the most important element for controlling gene's expression; it could drive gene express in special time, tissues and decide the level of expression. OsWRKY13 in rice is a pathogen inducible gene, including Xanthomonas oryzae pv. oryzae, causing bacterial blight disease, and Pyricularia grisea sacc., causing blast disease. Also it could be induced by hormone inoculation. Research on the promoter of OsWRKY13 will help us to explain the function of this gene and discover the plant disease resistance pathway. Another gene which encoding the 10 kD polypeptide in photosystemâ…¡has a tissue-specific expression manner. The analysis of gene's promoter region will help to tell the mechanism of tissue-specific expression and predict the function of this unknown protein.Using the eDNA clone which contains OsWRKYI3 gene as probe, we identified the subclone of genomic fragment contains the whole gene structure. After promoter prediction, we isolated a 728 bp promoter fragment, designated as P_OsWRKY13. We checked the expression mode in the transgenic plants and proved that the promoter could be induced by pathogen and hormone. In the analysis of 5'-end deletion mutants, gel retardation assay and site-directed mutation, we obtained the information about the cis-acting elements for responding to the pathogen inoculation. Using these elements as "bait", we gained the proteins which interact with these elements. After examine on the genes which encode these proteins, we found that these genes all have the pathogen inducible expression pattem and play roles in cell nuclear, these may indicate that these protein had function as transcription regulators.We also screened a cDNA clone which has a green-tissue but non-endosperm and embryo expression manner. Further we isolated the 2.1 kb promoter region and designated as P_D54O. Transgenic plants proved that it could drive the GUS reporter gene express in green-tissues. A series 5'-end deletion mutants have changed tissue-specific expression pattem and also the expression level, it implies that the promoter region contains cis-acting elements which control the tissue-specific expression and correspond for activating or suppressing gene expression. Hiring the gel retardation assay, we identified these elements and proved these elements using site-directed mutants. Utilize these elements as "bait", binding proteins are screened and the genes which encode the proteins are proved have different expression level in different tissues. The proteins also have cell nuclear localization and predicted to be transcription regulators.These promoters could be utilized in plants molecular breeding. It could be help to resolve the problem of transgenic safety. Also it could be the powerful tools to drive the foreign gene express in more efficient and pertinence way for the genetically modified plants. In the future, we could use the special elements we discovered to produce the special promoters for special use in production.