贝莱斯芽孢杆菌LJ02中植物免疫蛋白的筛选及其功能

Screening and Function of Plant Immune Proteins from Bacillus velezensis LJ02

【目的】贝莱斯芽孢杆菌(Bacillus velezensis)LJ02菌株能够诱导黄瓜等植物产生免疫抗病反应。本研究从生防菌LJ02中分离鉴定具有激发植物免疫活性的蛋白分子,通过验证其功能,进一步解析免疫蛋白的免疫信号通路。【方法】用硫酸铵沉淀法处理LJ02菌株发酵液,离心获得LJ02蛋白粗提物,将其经凝胶层析后再利用半制备高效液相色谱(high performance liquid chromatography,HPLC)分离,收集不同峰值处的蛋白组分。以烟草花叶病毒(tobacco mosaic virus,TMV)为靶标进行活性组分的筛选,从而获得具有免疫活性的组分。液相质谱联用仪(LC-MS)检测分析活性组分后发现,在组分F-23中含鞭毛蛋白(flagellin,Flg^(LJ02))。将鞭毛蛋白Flg^(LJ02)进行原核表达并纯化,经过敏性坏死反应(hypersensitive reaction,HR)和抗病试验验证其免疫功能。为确定其主要免疫信号通路,采用实时荧光定量PCR(qRT-PCR)检测水杨酸(salicylic acid,SA)和乙烯(ethylene,ET)途径的相关合成酶基因ICS1、PAL、ACS1以及免疫相关抗性基因NPR1、PR-1、EIN2和EIN3,通过免疫相关抗性基因的相对表达量解析鞭毛蛋白Flg^(LJ02)免疫信号转导途径。【结果】HPLC层析分离出贝莱斯芽孢杆菌LJ02菌液产生的60个外泌蛋白组分(F-1—F-60),免疫烟草接种TMV后,观察分析发现组分F-20、F-23、F-41、F-44具有显著的免疫抗病效应,其中组分F-23抗TMV效果最为显著,其抑制率为81.7%。经过质谱数据分析发现该组分包含鞭毛蛋白Flg^(LJ02)等7种物质。选择鞭毛蛋白Flg^(LJ02)为研究对象构建表达载体,转化大肠杆菌BL21,诱导表达后破碎细胞,获得粗提蛋白。将粗提蛋白过柱、透析纯化后的Flg^(LJ02)渗入珊西烟(Nicotiana tabacum var.xanthi)叶片,24 h后能观察到过敏性坏死反应。将Flg^(LJ02)稀释为终浓度50、100、200μg·mL^(-1)的稀释液后预处理珊西烟叶片,24 h后接种TMV,其对TMV的枯斑抑制率分别为65.6%、76.1%、88.1%。用鞭毛蛋白Flg^(LJ02)处理珊西烟,其SA途径和ET信号途径的免疫抗病相关基因ICS1、PAL、NPR1、PR-1、ACS1、EIN2、EIN3的相对表达量在24—48 h均显著上调。【结论】贝莱斯芽孢杆菌LJ02外泌的鞭毛蛋白Flg^(LJ02)可以激发珊西烟植株的免疫防御反应,提高烟草植株对TMV的抗病性。

【Objective】Bacillus velezensis LJ02 induces the immune response of cucumbers and other crops.The objective of this study is to screen and identify the immune proteins of LJ02,and further analyze the immune signalling pathways by verifying their functions.【Method】The LJ02 fermentation broth was precipitated with ammonium sulfate precipitation method,and the crude proteins of LJ02 were obtained by centrifugation.Then crude proteins were gel chromatographed and separated by high performance liquid chromatography(HPLC)to collect protein components at different peaks.The immune components were tested with tobacco mosaic virus(TMV)to obtain the immune components in plant.Liquid-phase mass spectrometry(LC-MS)detection and analysis revealed that the component F-23 contained flagellin(Flg^(LJ02)).Purified recombinant protein Flg^(LJ02)produced from Escherichia coli expression system was infiltrated into tobacco leaves and its immune function was verified by hypersensitive reaction(HR)and immune resistance analysis.Real-time fluorescent quantitative PCR(qRT-PCR)was used to detect salicylic acid(SA)and ethylene(ET)key synthetase genes ICS1,PAL,ACS1 and immune-related resistance genes NPR1,PR-1,EIN2 and EIN3.The relative expression of its immune-related resistance genes was tested to identify the Flg^(LJ02)-induced immune signal transduction pathway.【Result】Sixty exocrine protein components(from F-1 to F-60)of LJ02 were separated from HPLC,and components F-20,F-23,F-41,F-44 had strong immune effect against TMV in tobacco,among which,component F-23 showed the most significant anti-TMV effect,with an inhibition rate of 81.7%.Further mass spectrometry analysis found that this component contained flagellin Flg^(LJ02)and other 6 substances.The Flg^(LJ02)expression vector was constructed,and then transformed into E.coli BL21,the cells were broken after inducing the expression of Flg^(LJ02).The crude protein was eluted with Ni column purification,further dialyzed,then injected Flg^(LJ02)into tobacco leaves,hypersensitive reactions appeared about 24 h.Tobacco leaves was infiltrated with 50,100 and 200μg·mL^(-1) Flg^(LJ02),and then TMV was inoculated 24 hours post Flg^(LJ02)inoculation(hpi).The inhibition rate of Flg^(LJ02)against TMV was 65.6%,76.1%and 88.1%,respectively.The qRT-PCR determined that the expressions of SA and ET via defense-related genes ICS1,PAL,NPR1,PR-1,ACS1,EIN2,and EIN3 in tobacco plants were significantly up-regulated in 24-48 h.【Conclusion】The Flg^(LJ02)secreted by the B.velezensis LJ02 activates the SA and ET signalling immune pathway,thereby improving plants disease resistance to TMV.