| In response to the pathogens attack or chemical inducers treatment, plant can activate physiological defense mechanism that render limitation to pathogens and subsequent infection, Resistance induction is accompanied by the activation of a set of self-defensive genes involved in formation of chemical barriers. Among them, PRs (pathogenesis-related proteins) genes are the crucial one, and serve as important marker genes for defense activation. Benzothiadiazole (BTH) is an inducer of SAR (systemic aquired resistance) in rice and enchances the resistance to blast disease. In this study, we cloned two BTH-induced rice PR genes OsPR-4a and OsGlu, respectively. Possible roles of OsPR-4a in rice SAR and defense against Magnaporthe grisea are discussed.A cDNA clone,bnhn-n 18,encoding a putative PR-4 protein was selected from the BTH induced rice SSH(suppressed substractive hybridization) cDNA library. With the BTH-induced rice full-length cDNA library DNA as a template, the cDNA end of OsPR-4a was cloned by PCR with the gene specific primer and a vector universal primer. The assembled full-length cDNA for OsPR-4a is 739 bp, encoding a 151 amino acid protein with a predicted molecular mass of 16.47 kD and p/ of 4.42.The deduced protein has a main domain Barwin, and belongs to the Barwin family. Similar to other members of the family, the deduced OsPR-4a has a conserved pattern of six cysteines involved in 3 disulfide bonds, which distinguishBarwin family from others. Using the primers corresponding to the cDNA untranslating region, the OsPR-4a genomic sequence was cloned by PCR. The OsPR-4a genomic sequence include an intron with 944bp in length. Sourthern blot analysis showed OsPR-4a integrate in rice genome with a single copy.Expression of OsPR-4a in interaction of rice isogenic lines H8S (susceptible) and HSR (resistant) with Magnaporthe grisea was analyzed through Northern blot. The expression ofOsPR-4a was detected 36 h after inoculation in incompatible but not in compatible interaction at any testing time. Northern blot also showed that expression of OsPR-4a was detected 24 h after treatment with 0.3 mmol/L BTH, but was not detected in the water treatment during 48 hrs after treatment in seedlings of susceptible cultivar. The dynamic expression of OsPR-4a in susceptible rice seedlings was analyzed by RT-PCR. The highest expression of OsPR-4a was detected at 36 h after inoculation; When the seedlings pretreated with 0.3 mmol/L BTH were challenged with M.grisea 3d after the treatment, the highest expression of OsPR-4a was detected by RT-PCR at 36 h and retained to 72 h after inoculation. These results demonstrate that expression of OsPR-4a can be induced by SAR inducer BTH or blast pathogen. After BTH treatment, expression of OsPR-4a can be intensified upon subsequent challenge infection by M. grisea.The cloned OsPR-4a was expressed in Escherichia coli and purified protein show antifungal activity against rice pathogens M. grisea and Rhizoctonia solani.A promoter sequence with 2257 bp upstream of the rice OsPR-4a was amplified using two primers designed based on sequence of OsPR-4a and rice genome sequences database. Several cw-acting elements, including W-box, PAL-boxA, GT-1 elements and MybStl elements, were recognized by PLACE program. The promoter region sequence and its 5' deletion constructs were fused to the gus reporter gene in pCAMBIA1391z vector for analyzing the promoter activity and the functions of the cis-acting elements in regulation of OsPR-4a expression.A cDNA clone,bnhn-n10,encoding a putative -1, 3-glucanase was selected from the BTH induced rice SSH cDNA library. With the BTH-induced rice full-length cDNA library DNA as a template, the 5' cDNA fragment of OsGlu was cloned by PCR with the gene specific primer and a vector universal primer. The assembled full-length cDNA for OsGlu is 2034 bp, encoding a 470 amino acid protein with a predicted molecular mass of 50.3 kD and p/ of 5.77. Deduced OsGlu protein is homologous to plant -1, 3-glucanase and belongs to Glycosyl... |
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