Cloning and characterization of an harpin-encoding gene from Xanthomonas axonopodis pv. glycines required for hypersensitive response on nonhost plant tobacco
大豆斑疹病菌harpin编码基因的克隆与特性研究

An hpa1 gene was cloned into an expression vector, pET30a(+), from the genomic DNA of Xanthomonas axonopodis pv. glycines (Xag), the causal agent of soybean bacterial pustule, with degenerated primers by polymerase amplification reaction (PCR). The gene product was extracted from the conjugate (BHR-3) of BL21 (DES) with the recombined vector pHR3 after the engineering strain was induced by IPTG in LB medium. The SDS-PAGE gel showed that the gene product was 15.1kD. The product was heat-stable (10 min at 100 degrees C), protease K sensitive, and able to trigger hypersensitive response (HR) in common tobacco, but was unable to elicit HR in NahG transgenic tobacco in which salicylic acid accumulation was abolished. Moreover, the HR elicitation of the protein in tobacco was dispelled by eukayotic metabolic inhibitors, actinomycin D, cycloheximide and LaCl3. The 402 bp hpa1 gene in this study putatively encoded a 133 ammonia acid protein of which glycine (G) was rich with 21.1%. Sequence comparison indicated that the hpa1 gene and its protein was 51.4% - 93.8% identity with those of Xanthomonas oryzae pv. oryzae and other Xanthomonas species and pathovars. Alignments of harpin proteins of Xanthomonas genus displayed that the glycine-rich region with GGG-GG motif was variable. The comparison also showed that the harpin-encoding gene of Xag (nominated here as hpa1(Xag)) did not possess any similarity with that of Erwinia amylovora, Pseudomonas syringae and Ralstonia solanacearum at nucleotide and protein levels. It is concluded that hpa1(Xag) gene encodes an harpin protein which elicits a typical HR in nonhost tobacco.