Analysis Of Rice Pr Gene Promoters And Their Regulation In Response To Rice Bacterial Blight Disease

Pathogenesis-related (PR) genes are induced by pathogen attack or exogenous hormones treatment and have been widely used as a marker for systemic acquired resistance (SAR). Transcription initiation plays a key role in the regulation of gene expression. The specific promoter of an initiation process can decide whether it be expressed or the expression level of the gene. In order to gain a further insight into the expression/regulation of marker gene OsPR1b, OsPR5and OsPR10a in rice immune response, expression analysis of the three genes in rice was carried out. We used GUS reporter gene for histochemical GUS staining analysis and GUS activity analysis. In addition, the expression level of OsPR1b gene was analyzed in the rice leaves and roots respectively treated with plant hormones, three abiotic factors and Xanthomonas oryzae pv. oryzae(Xoo) PXO124strain. The main content of the experimental study are as follows:1. Promoter fragments of three PR genes were amplified from rice Nipponbare genome and named OsPRlbp, OsPRSp, OsPR10ap. We forecast several putative stress-responsive cis-elements in OsPR1bp, OsPR5p and OsPR10ap from PLACE website. The three Promoter fragments were fused with GUS gene to construct, and plant expression vectors were constructed respectively. Then the vectors were introduced into japonica rice via Agrobacterium-mediated transformation.2. Real-time PCR analysis showed the expression level of OsPR1b gene was much higher in leaf than that in stem, root, callus and flower. Similarly, OsPR5also showed a higher expression level in leaf. While OsPR10a exhibited a strong expression in various tissues.3. OsPR1bp::GUS and OsPR5p::GUS activity can only be observed in leaves when in seedling stage, while different levels of GUS activity can be detected in various tissues of OsPR10ap::GUS plants. In OsPR10ap::GUS plants, the GUS activity in root mainly located in apical meristem area and lateral root. GUS staining in young leaves mostly concentrated in tip and margin. The pattern of leaves became inhomogeneous stained with scattered spots in seedling stage and blossom stage. OsPR5p::GUS showed significant wounding response in cross cut of petioles and flag leaves.In addition, GUS activity was observed in leaf hairs of OsPR10ap. OsPR1bp had no expression in flowers, while OsPR5p and OsPR10ap expressed in glumes and pollens. All of the three promoters had expression in the embryos of germinating seeds. OsPRlbp had weak expression compared with the strong expressions of OsPR5p and OsPR10ap.4. We analyzed inducible expression of the three PR genes in wild type rice, the results showed that:the expression level of OsPR1b in leaves can be increased by SA, MeJA, KT, ABA, NaCl and PEG, and its expression level in root can be enhanced by MeJA, KT and NaCl. The treatments of MeJA, GA, KT, ABA, NaCl and H2O2can increase the expression level of OsPR5in leaves, and its expression level in root can be enhanced by MeJA, KT, ET, NaCl, PEG and H2O2. The Expression level of OsPR10a in leaves can be increased by MeJA, KT, ABA, NaCl and PEG. However, expression quantity of the genes in leaves and roots were different under these hormones treatments. Inoculation with P10alone for24hr had no effect on gene expression, while co-treated with MeJA could significantly enhanced the expression level of OsPR1b and OsPR5in leaves.Furthermore, to get a further insight of the regulation of PR genes expression in the hormone signaling pathway(s) mutants and the role of plant hormones in rice immune regulation, we cloned two SAMT genes and constructed the vectors of over-expression, gene silencing, subcellular localization and promoter analysis, then introduced them into japonica rice for further study.