Identif ication of a hypersensitive response core peptide of HrpZ and its role in increasing grape downy mildew resistance

Harpins play a key role in inducing disease resistance in crops,and identifying their core functional regions and establishing a system for their efficient expression would be very valuable.In this study,large amounts of soluble fusion proteins of harpin HrpZ and its subpeptides were obtained via the optimized induction conditions(28℃ with 0.5 mmol·L^(-1) IPTG for 6 h)in Escherichia coli BL21(DE3).Hypersensitive response(HR)assays demonstrated that the C-terminal 66 aa of HrpZ(HrpZ_C_2_2)elicited a strong HR in tobacco(Nicotiana benthamiana)and grape(Flame Seedless)leaves.Additionally,treatment with HrpZ,and particularly HrpZ_C_2_2,significantly reduced the disease incidence and severity index of field vine leaves and those inoculated with downy mildew.The determination of the physiological parameters indicated that HrpZ,and especially HrpZ_C_2_2,improved the photosynthesis-and chlorophyll fluorescence-related parameters,enhanced the activity of defense-related enzymes,including SOD,POD,CAT and PAL,and increased the H_(2)O_(2) level.Collectively,we efficiently expressed a core peptide of HrpZ and elucidated its strong ability to elicit a HR and resistance to downy mildew.This research provides insight into understanding the structure and function of HrpZ and will advance the application of HrpZ_C_2_2 to increase the resistance of grapevine to downy mildew.

烟草野火病菌hrpZ基因的克隆及蛋白的原核表达

采用PCR方法从烟草野火病病原菌Psta218中扩增harpin编码基因hrpZ,将其克隆到原核表达载体pGEX-4T-1上,获得重组表达质粒pGEX-hrpZPsta。将重组质粒pGEX-hrpZPsta转化至大肠杆菌BL21(DE3)菌株,得到重组大肠杆菌BL21/pGEX-hrpZPsta。经IPTG诱导得到14.7 kD的蛋白质。该蛋白质与其他已发现的harpins一样,能够使烟草等植物产生过敏性坏死反应、富含甘氨酸、热稳定以及对蛋白酶K敏感。

HrpZ激发对几种植物防御相关酶活性的影响

[目的]测定HrpZ激发对黄瓜、番茄和茄子3种防御酶活性的影响情况。[方法]以盆栽黄瓜、番茄和茄子幼苗为材料,研究了15μg/ml的HrpZ激发黄瓜、番茄和茄子3种植株幼叶后,不同时间苯丙氨酸解氨酶(PAL)、过氧化物酶(POD)、多酚氧化酶(PPO)3种防御酶活性的影响变化。[结果]经HrpZ激发后,植株叶片中的3种防御酶活性均有不同程度的提高。HrpZ喷施黄瓜12、24和48 h后,PPO、PAL、POD活性相应比清水对照提高83.0%、54.1%和39.3%;HrpZ喷施番茄12、24和48 h后,POD、PPO、PAL活性相应比清水对照提高31.8%、44.6%和33.3%;HrpZ喷施茄子1和3 h,POD、PPO活性相应比清水对照提高228.4%和142.5%。[结论]HrpZ能诱导植物PAL、POD、PPO的表达,从而诱导植物防御反应和系统获得抗性,增强植物的抗病性。

hrpZ基因在大肠杆菌中的高效表达与活性检测

PCR扩增hrpZ基因片段，克隆到pGEM-T载体中，经EmR Ⅰ／Xho Ⅰ酶切、连接将hrpZ基因插入大肠杆菌表达载体pET32b(+)硫氧还蛋白下游，构建重组表达质粒pET-hrpZ，再将其转化至E．coli BL21(DE3)中，经筛选得到阳性克隆子，IPTG诱导表达HrpZ蛋白。SDS-PAGE显示，目的蛋白在重组菌株中得到了可溶性高效表达。该重组蛋白分子量为55．8 kD，与理论值大小相符。采用叶片穿刺法，融合蛋白具有诱导烟草过敏反应的生物功能。