1. The Hypersensitive Response (HR) Of Rice Cells Induced By Signal Moleculars 2. Cloning A Chitinase Gene From A New Isolated Pathogenic Bacterium Strain For Spodoptera Exigua Hüber And Expression The Gene In Bacillus Subtilis

The hypersensitive response (HR) is one of a key feature of plant disease resistance. To study the effects and mechanisms of signal molecular, such as NO and H₂O₂ in the HR induction of rice cells, will be helpful to understand the defense response mechanisms and signal process of rice cells.Solutions presented in this study can be summarized as follows:1. The establishment and optimization of quantitative measurements of hypersensitive response (HR) in the cultured rice cells was reported. Bromphenol blue is a more suitable and sensitive dye used for the quantitative measurements of hypersensitive response (HR) of rice cells than Evan's blue. Linear regression analysis reveals there is a close correlation (0.9994) between the values of maximum absorbance and the concentrations of Bromphonel blue arranged from 0.625μg·mL^-1 to 20μg·mL^-1. The optimum measurement steps are as follows: Rice cells staining for 15 min with 0.03% Bromphenol blue, washing with the running water to remove the unbound dye, dissolving the bound dye with the solutions of 50% methanol and 1% SDS at 50℃for 30min, and then measure the absorbance of the extracts at 595 nm, with those of the methanol-treated rice cells for 15min as a control of 100% cell death. The percentage of rice death cells assayed with Bromphenol blue under the above optimum conditions is consistent with the actual one.2. Hypersensitive response (HR) of rice cells induced by hydrogen peroxide (H₂O₂), a signal molecule, was investigated. Addition of glucose and glucose oxidase as the H₂O₂-generating system to rice suspension cells resulted in a steady-state production of H₂O₂ and induction of HR. However, exogenously applied H₂O₂ was completely degraded by rice cells and no HR was observed. H₂O₂ generated from the H₂O₂-generating system could be scavenged by the supplement of catalase. These results indicated that H₂O₂-induced HR in cultured rice cells occurred in a time-dependent manner. Furthermore, the production of H₂O₂ was involved in an incompatible bacterial interaction.3. The hypersensitive response (HR) in rice suspension cultured cells induced by exogenous addition of the signaling molecules, nitric oxide (NO) and hydrogen peroxide (H₂O₂) was studied. Results showed that HR of rice cells could be induced by NO and H₂O₂ dependently and/or in synergy and that NO-induced HR could be regulated by H₂O₂. The balance interaction in concentrations between NO and H₂O₂ was not found in the induction of rice cell deaths.To elucidate the nature of rice susceptible response to infection by Xanthomonas oryzae pv. oryzae (Xoo) at the genomic levels, analysis of the cDNA amplified fragment length polymorphism (AFLP) pattern of mock- and Xoo-inoculated rice suspension cultured cells was performed. Three hundred and sixty-three (9.1%) from approximately 4,000 cDNA fragments analyzed were differentially regulated (295 up-regulated and 68 down-regulated) 0.5, 3, 24 h after co-cultivation with Xoo. Seven types of gene expression patterns of the differential fragments were identified. Ten of 31 sequenced fragments showed homology to genes with known or putative functions involved in metabolism, pathogen response and signaling, while 21 others did not show any homology to sequences with known functions, cDNA-AFLP differential patterns for 5 selected genes were confirmed via real-time reverse transcription-PCR analysis. This work begins to reveal potential disease susceptibility-related genes of this staple crop with global importance.For the purpose of construction the genetic engineering bacterium to control the crops dangerous pest of Spodoptera exigua Huber. Pathogenic bacterium strain was isolated and identified from the pathogenic larvae of Spodoptera exigua Hunber. A chitinase A gene (chiA) was successfully cloned from the strain, then the gene was inserted in a expression vector and expressed in Bacillus subtilis strains in high levels.Solutions presented in this study can be summarized as follows:1. Pathogenic bacterium strain QL-1 was isolated from the pathogenic larvae of Spodoptera exigua Hunber and the physiological characters of the strain were studied. The results show that the physiological characters of the isolated QL-1 strain are similar to the Serratia marcescens species, which belongs to the genus Serratia, a member of the Enterobacteriaceae. The 16S rDNA sequence for the PCR amplified fragment of the strain was also analyzed, and then a phylogenetic tree was constructed by comparing with the published 16S rDNA sequences of the relative bacteria species. In the phylogenetic tree QL-1 was the closest relative to Serratia marcescens with more than 99.2% sequence similarity.2. A chitinase A gene (chiA) was successfully amplified by PCR using a pair of specific primers from Serratia marcescens Q901, and cloned into pUC19 vector. Nucleotide sequence analysis indicated that chiA sequence (GenBank Accession Number: AY433954) with a length of 1,841bp included an open reading frame (ORF) of 1,692bp, which encoded 563 amino acids. Analysis of amino acid sequence homology showed that ChiA is a number of 18 family in glycosyl hydrolase and contains a signal peptide (23 amino acid residues) and two functional domains: a PKD domain (73 amino acid residues) and a catalytic domain (387 amino acid residues).3. The cloned chitinase A gene (chiA) was inserted into the shuttle vector of pMA5, the antibiotic resistance gene of Ampr in the construction vector was excised, yielding the expression plasmid of pMA5-chiA-2, and then the pMA5-chiA-2 plasmid was transformed into Bacillus subtilis strain BS168, BCL1050. High levels of chitinase were produced in the transformed strain.