摘要
为探究枸杞内生嗜线虫镰刀菌NQ8GII4(Fusarium nematophilum)分泌的效应蛋白FnE14152的功能,本研究分析了该蛋白的结构特征,利用逆转录PCR(RT-PCR)法克隆了NQ8GⅡ4菌株效应蛋白编码基因FnE14152,并将该编码基因构建到pET30a载体中,重组质粒转化至大肠杆菌(Escherichia coli)BL21(DE3)表达菌株。采用不同浓度的异丙基-β-D-硫代半乳糖苷(IPTG)在不同诱导时间及温度下诱导表达重组蛋白,十二烷基硫酸钠聚丙烯酰胺凝胶电泳(SDS-PAGE)检测上清及沉淀中蛋白的表达。通过镍柱亲和层析法纯化蛋白并进行蛋白质免疫印迹(Western blot)鉴定,进一步将纯化的目的蛋白注射到烟草叶片中。生物信息分析结果表明,该基因编码274个氨基酸,分子量为29.04 kD,等电点为4.58,平均亲水值为-0.043,是偏亲水蛋白质,推测为糖苷水解酶16家族(glycoside hydrolase 16,GH16)转糖基化酶。成功克隆得到长度为774 bp的去信号肽效应蛋白FnE14152基因,将该基因构建到原核表达载体pET30a-14152^(ΔSP),经0.2 mmol·L^(-1)IPTG诱导得到重组蛋白pET30a-14152^(ΔSP);在20℃条件下培养13 h时,上清中的蛋白表达量最高,灰度分析结果极显著。生测试验结果表明重组蛋白注射4 d后可以诱发烟草叶片组织产生细胞坏死反应。本研究获得了枸杞内生嗜线虫镰刀菌NQ8GⅡ4效应蛋白FnE14152,为进一步明确内生真菌效应蛋白诱发植物的免疫机制奠定了基础。
To investigate the function of the effector protein FnE14152 secreted by the endophytic Fusarium nematophilum strain NQ8GII4 of Lycium barbarum,its encoding gene FnE14152 was cloned with reverse transcription PCR(RT-PCR),and its structural characteristics were analyzed.FnE14152 was amplified and ligated into the pET30a vector.The recombinant vector was transformed into the Escherichia coli BL21(DE3)expression strain.After optimizing IPTG concentrations,induction times and temperatures,the expression of FnE14152 was detected by SDS-PAGE.The protein was purified using nickel-affinity chromatography and identified by Western blot.Purified FnE14152 was injected into Nicotiana benthamiana leaves.Results showed that FnE14152 encodes 274 amino acids with a molecular weight of 29.04 kDa,an isoelectric point of 4.58,and an average hydrophilic value of-0.043.It is a hydrophilic,bias protein predicted to function as a glycoside hydrolase 16(GH16)transglycosylation.The recombinant vector pET30a-FnE14152^(ΔSP)(lacking the signal peptide,FnE14152^(ΔSP))was constructed.Optimal production of soluble recombinant protein were occurred when the transformed strain was induced with 0.2 mmol·L^(-1)IPTG and culture at 20℃for 13 h,yielding the highest FnE14152 abundance in the supernatant,as confirmed by grayscale analysis.The recombinant protein induced necrotic in tobacco leaf 4 days post-injection.In this study,FnE14152 were obtained from endophytic Fusarium nematophilum strain NQ8GII4,which lays a foundation for further clarification of the immune mechanism induced by endophytic fungal effector proteins in plants.
作者
郭苗苗
闫思远
张强强
顾沛雯
GUO Miaomiao;YAN Siyuan;ZHANG Qiangqiang;GU Peiwen(School of Agriculture,Ningxia University,Yinchuan,Ningxia 750021;College of Forestry and Prataculture,Ningxia University,Yinchuan,Ningxia 750021)
出处
《核农学报》
北大核心
2025年第9期1896-1905,共10页
Journal of Nuclear Agricultural Sciences
基金
国家自然科学基金(32260697)。
关键词
枸杞内生嗜线虫镰刀菌
效应蛋白FnE14152
原核表达
条件优化
细胞坏死反应
endophytic Fusarium nematophilum of Lycium chinense
effector protein FnE14152
prokaryotic expression
conditional optimization
cell death reaction
