大豆斑疹病菌harpin编码基因的克隆与特性研究

根据黄单胞菌harpin编码基因的同源性，设计简并引物，采用PCR方法从大豆斑疹病菌（Xanthomonas axonopodis pv.glycines, Xag）中克隆了402 bp的STBX]hpa1STBZ]同源基因，构建于表达载体pET30(a)上经转化大肠杆菌BL21菌株，获得基因工程菌BHR_3。基因工程菌诱导表达后经收集菌体和破碎细胞，得到表达产物为151kD的蛋白质。该蛋白质富含甘氨酸，不含半胱氨酸，对热稳定，对蛋白酶K敏感，可在非寄主烟草上激发过敏反应。激发的过敏反应需要植物体内水杨酸的积累，还可被真核生物代谢抑制剂抑制。序列比较显示，该基因与Xag中hpaG基因相同，与其它黄单胞菌中的hpa1基因有51.4%～93.8%的同源性，与其它革兰氏阴性植物病原细菌的harpin编码基因无同源性。据此把该基因产物鉴定为harpinXag。黄单胞菌harpin蛋白质序列比较发现，GG_GGG基序的多少并不是harpin蛋白的唯一特性。这为利用harpin蛋白开展植物病害控制的基因药物学设计提供了科学线索。

Cloning and characterization of an harpin-encoding gene from Xanthomonas axonopodis pv. glycines required for hypersensitive response on nonhost plant tobacco

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.