新型水稻黄单胞菌Harpin蛋白的纯化及其特性研究

报道水稻黄单胞(Xanthomonas oryzae)的两个致病变种(pv.oryzae和 pv.oryzicola)所产生的Harpin蛋白. 结果表明， 表达菌株HRF1和HRF2分别携带两个致病变种编码Harpin的hrf1和hrf2基因. 在IPTG诱导下, 两个表达菌株的无细胞破碎液均具有激发烟草叶片过敏反应(HR)的活性. 采用(NH4)2SO4沉淀、 阴离子交换层析、 Native-PAGE微量制备等方法, 分别纯化出分子量为15 600和15 300, pI皆为4.5左右的单一条带; 这两个单一组分符合典型Harpin蛋白的特征: 可激发烟草HR， 诱导烟草抗TMV， 对蛋白酶K敏感、 对热稳定; 放线菌素D、 环己酰亚胺和氯化镧等真核生物代谢抑制剂可消除它们的生物活性; 琼脂双扩散试验(ODD)血清反应表明， 两个Harpin蛋白有交叉反应.

Purification and Characterization of a New Type of Harpins from Xanthomonas oryzae Pathovars

The Harpins produced by two pathovars(pv. oryzae and pv. oryzicola) of Xanthomonas oryzae are different from the reported harpins. The recombinant E.coli strains HRF1 and HRF2 harbored hrf1 gene of pv. oryzae and hrf2 gene of pv. oryzicola, respectively. After induced by IPTG, the Cell Free Crude Solution(CFCS) of hrf1 and hrf2 can elicit hypersensitive reaction(HR) on tobacco leaves. Precipitated CFCS with(NH4)2SO4(30%—60%), purified with HiPrep Q XL ion exchange and Mini prepared Native PAGE(10%), two single bands with molecular weights of 15 600 and 15 300, and about pI=4.5 were respectively obtained. The results show the same characteristics to other Harpins: elicit HR, induce TMV resistance on tobacco leaves, sensitive to protease K and stable to heat. Their biological activity can be inhibited by eucaryota metabolic inhibitors(cycloheximide, actinomycin D and LaCl3). Meanwhile, serological experiments of the proteins with ODD indicate that cross reactions have occurred in two Harpins from pv.oryzae and pv.oryzicola.