Xenorhabdus budapestensis D43菌株杀虫蛋白的分离纯化及致病机理的研究
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摘要
昆虫病原线虫共生菌是一种重要的微生物资源，它能够产生多种具有杀虫、抑菌、抗肿瘤等功能的代谢产物，在农业和医药卫生领域显示出巨大的开发潜力和应用前景。特别是昆虫病原线虫共生菌能够产生多种杀虫蛋白，不仅可以直接作为蛋白质农药，而且能够为转基因抗虫植物和杀虫工程菌株的发展提供更多新颖的基因资源。本研究以从中国东北土壤样品获得的拟双角斯氏线虫中分离的共生菌Xenorhabdusbudapestensis D43为试验材料，对该菌株细胞内的蛋白进行了纯化和筛选，并对筛选到具有杀虫活性的蛋白进行了鉴定、基因克隆、序列生物信息学分析、重组蛋白的表达和纯化，以及致病机理等方面的研究，得出主要研究结果如下：
1、昆虫病原线虫共生菌X. budapestensis D43菌株细胞内含有大量的蛋白种类，本研究利用制备型凝胶电泳的方法成功纯化到4种，其中一种对大蜡螟幼虫具有血腔注射活性，命名为HIP57。生物测定结果显示，该蛋白对5龄大蜡螟幼虫的LD50为206.81ng/头；当以不低于490ng/头的剂量注射时，试虫体色会在15min之内变黑，48h死亡率为100%。
2、经过双向电泳分析和质谱鉴定，HIP57的分子量约为60kDa，等电点4~5，与GroEL蛋白具有较高同源性。GroEL虽为分子伴侣，但在本研究中，来自于共生菌X.budapestensis D43菌株的HIP57显示出杀虫功能。克隆获得HIP57基因全长1647bp（GenBank accession no. JN863588），编码548个氨基酸残基（GenBank accession no.AEU10771）。
3、生物信息学分析结果显示，HIP57蛋白的相对分子量为57379.7Da，理论等电点为4.77，与电泳结果相吻合；该蛋白序列中不含信号肽，属于稳定的水溶性蛋白；同时采用4种不同的预测原理分析了HIP57的二级结构，特别是运用同源模建构建了精确度较高的HIP57蛋白三级结构；通过HIP57蛋白与其他4种同样来源于昆虫病原线虫共生菌的GroEL蛋白进行序列比对发现，该蛋白拥有一些特有的氨基酸残基；根据GroEL蛋白氨基酸序列的同源性，本研究成功构建了43种肠杆菌科细菌的系统发育树，将X. budapestensis D43聚类至致病杆菌属，显示出GroEL蛋白序列具有在细菌分类学和进化学应用的潜在性。
4、对HIP57、GFP和HIP57-GFP进行了原核表达，同时获得了纯度较高的3种重组蛋白。经western blotting分析，这些重组蛋白与其相应的抗体都能够发生特异性结合。与天然HIP57蛋白引起试虫通体黑化现象和高致死活性相比，HIP57和GFP重组蛋白没有显示出这样的功能；而HIP57-GFP重组蛋白虽不对试虫具有致死活性，但是能够引起试虫体内出现大量的黑色结节。
5、HIP57蛋白对大蜡螟幼虫致病机理的研究结果包括：其一，HIP57能够引起大蜡螟幼虫强烈的免疫反应，包括PO活力显著升高，血细胞数量显著降低，以及血细胞包囊反应；其二，大蜡螟注射HIP57后，有23种血淋巴蛋白的表达量发生显著变化，其中4种获得成功鉴定，分别为ApoLp-III、Serpin、P27K和GST；其三，捕获2种与HIP57具有潜在结合作用的血淋巴蛋白，分别为芳基贮存蛋白和β-1，3-葡聚糖识别蛋白前体。
虽然在昆虫病原线虫共生菌复合体侵染寄主昆虫过程中，HIP57是否被分泌的问题本研究未能给出答案，但HIP57显示出非常高的致病性，如果在昆虫病原线虫共生菌复合体致病过程中能够被分泌到寄主血腔中，应为一种重要的致病因子；即便在线虫共生菌致病过程中不能分泌到寄主血腔，HIP57作为一种拥有较高致病性的蛋白，其研究价值和应用潜力仍然十分值得关注。本研究结果不仅为揭示HIP57蛋白的杀虫机理奠定了基础，而且为该蛋白的合理利用提供了理论依据。
The entomopathogenic bacteria, symbiotically associate with different families ofnematodes, has been described to produce a range of metabolites that have insecticidal,antibacterial，anticancer activity, and so on. At present, these bacteria have been regardedas an important microbial resource and have huge development potential and goodapplication prospect. In particular, entomopathogenic bacteria can produce a range ofnovel toxin proteins that could be directly applied as protein insecticides or used in thetransgenic plants and microbes. Xenorhabdus budapestensis D43live in symbiosis withentomopathogenic nematode Steinernema ceratophorum, which was isolated fromnortheast China soils by using the insect bait entrapment method. In this study, we directlypurified new intracellular protein fractions from X. budapestensis D43that were screenedby insect bioassay. Subsequently, new insecticidal protein was studied in various aspectsinclude protein identification, gene cloning, bioinformatic analysis, recombinant proteinexpression and purification, and pathogenetic mechanism. The main results of this studycan be summarized as follows:
1. In this report, we purified four protein fractions from the intracellular extract of X.budapestensis D43by preparative native PAGE. However, only one protein exhibitedinsecticidal activity against Galleria mellonella larvae by injection that was designatedHIP57. By injection, HIP57caused G. mellonella larval bodies to blacken and die with anLD50of206.81ng/larva. Especially, when we injected no less than490ng of HIP57perlarva, almost all G. mellonella larvae developed blackened body color within15min anddead after forty-eight hours.
2. HIP57has a molecular weight (MW) of57379.7Da and a theoretical isoelectric point(pI) of4.77. The full-length1647bp sequence of hip57was obtained by sequencing and itssequence has been deposited in GenBank (accession no. JN863588), whereas its deducedamino acid sequence of548amino acids was deposited as AEU10771(GenBank accessionno.). HIP57protein is homologous to GroEL proteins from other bacteria. GroEL has beenaccepted as molecule chaperon, however, our research revealed that HIP57(GroEL)possesses another novel function as an insecticide in X. budapestensis D43.
3. A bioinformatic analysis suggested that signal peptide is absent in HIP57protein, whichis stable and soluble in water. HIP57secondary structure prediction has been performed byfour different methods. At the same time, we predicted the accurate three-dimensional structure of HIP57with high accuracy by homology modeling. By multiple sequencealignment, we found that HIP57contained several specific amino acid residues, which didnot appear in any of the other four GroEL proteins from the family of mutualistic bacteriaat the same positions. A phylogenetic tree was constructed base on the homologues offorty-three GroEL proteins from the family of Enterobacteriaceae. In the phylogenetic tree,X. budapestensis D43were successfully classified into Xenorhabdus, which indicated thatGroEL have potential application in bacterial taxonomy and evolution.
4. The recombinant HIP57, GFP and HIP57-GFP proteins were respectively expressed andpurified from transformed E. coli. Western blotting analysis show that these recombinantproteins could bind specifically to antibodies, respectively. Compared with the case ofoccurring melanism involve darkening of the whole body of wax moth larvae injected withnatural HIP57protein, recombinant HIP57protein did not show similar virulence.However, HIP57-GFP recombinant proteins was enable for the formation of black nodulein the haemocoel of larvae, but not lethal.
5. G. mellonella larvae were injected with HIP57, resulting in extensive immune responsesinclude a significant increase in PO activity, a significant decline in hemocyte populationsand encapsulation response of hemocytes, compared to the controls. Our results illustratepronounced changes in the expression of four genes encoding hemolymph proteins,including ApoLp-III、Serpin、P27K and GST, were observed after HIP57exposure. Weinvestigated the hemolymph proteins that interact with HIP57protein usingco-immunoprecipitation. In vitro two interactions were arylphorin and β-1,3-glucanrecognition protein precursor, which were identified.
In this research we unsuccessfully answer the problem that is if HIP57was secreted byentomopathogenic nematode-bacteria complex during infection host insect. However,HIP57show high lethal potency in the G. mellonella larva bioassay, thereby hypothesizingthat the bacteria could secret HIP57, which may play an important role inentomopathogenic nematode-bacteria complex infecting host insect. Even if the abovehypothesis is not confirmed, HIP57has research significance and application potency as aresult of excellent pathogenicity and novel insecticide mode. The results of my study forPh.D. degree are of greatly importance to elucidate the pathogenetic mechanism of HIP57,which will be useful for appropriate application of HIP57.
1、昆虫病原线虫共生菌X. budapestensis D43菌株细胞内含有大量的蛋白种类，本研究利用制备型凝胶电泳的方法成功纯化到4种，其中一种对大蜡螟幼虫具有血腔注射活性，命名为HIP57。生物测定结果显示，该蛋白对5龄大蜡螟幼虫的LD50为206.81ng/头；当以不低于490ng/头的剂量注射时，试虫体色会在15min之内变黑，48h死亡率为100%。
2、经过双向电泳分析和质谱鉴定，HIP57的分子量约为60kDa，等电点4~5，与GroEL蛋白具有较高同源性。GroEL虽为分子伴侣，但在本研究中，来自于共生菌X.budapestensis D43菌株的HIP57显示出杀虫功能。克隆获得HIP57基因全长1647bp（GenBank accession no. JN863588），编码548个氨基酸残基（GenBank accession no.AEU10771）。
3、生物信息学分析结果显示，HIP57蛋白的相对分子量为57379.7Da，理论等电点为4.77，与电泳结果相吻合；该蛋白序列中不含信号肽，属于稳定的水溶性蛋白；同时采用4种不同的预测原理分析了HIP57的二级结构，特别是运用同源模建构建了精确度较高的HIP57蛋白三级结构；通过HIP57蛋白与其他4种同样来源于昆虫病原线虫共生菌的GroEL蛋白进行序列比对发现，该蛋白拥有一些特有的氨基酸残基；根据GroEL蛋白氨基酸序列的同源性，本研究成功构建了43种肠杆菌科细菌的系统发育树，将X. budapestensis D43聚类至致病杆菌属，显示出GroEL蛋白序列具有在细菌分类学和进化学应用的潜在性。
4、对HIP57、GFP和HIP57-GFP进行了原核表达，同时获得了纯度较高的3种重组蛋白。经western blotting分析，这些重组蛋白与其相应的抗体都能够发生特异性结合。与天然HIP57蛋白引起试虫通体黑化现象和高致死活性相比，HIP57和GFP重组蛋白没有显示出这样的功能；而HIP57-GFP重组蛋白虽不对试虫具有致死活性，但是能够引起试虫体内出现大量的黑色结节。
5、HIP57蛋白对大蜡螟幼虫致病机理的研究结果包括：其一，HIP57能够引起大蜡螟幼虫强烈的免疫反应，包括PO活力显著升高，血细胞数量显著降低，以及血细胞包囊反应；其二，大蜡螟注射HIP57后，有23种血淋巴蛋白的表达量发生显著变化，其中4种获得成功鉴定，分别为ApoLp-III、Serpin、P27K和GST；其三，捕获2种与HIP57具有潜在结合作用的血淋巴蛋白，分别为芳基贮存蛋白和β-1，3-葡聚糖识别蛋白前体。
虽然在昆虫病原线虫共生菌复合体侵染寄主昆虫过程中，HIP57是否被分泌的问题本研究未能给出答案，但HIP57显示出非常高的致病性，如果在昆虫病原线虫共生菌复合体致病过程中能够被分泌到寄主血腔中，应为一种重要的致病因子；即便在线虫共生菌致病过程中不能分泌到寄主血腔，HIP57作为一种拥有较高致病性的蛋白，其研究价值和应用潜力仍然十分值得关注。本研究结果不仅为揭示HIP57蛋白的杀虫机理奠定了基础，而且为该蛋白的合理利用提供了理论依据。
The entomopathogenic bacteria, symbiotically associate with different families ofnematodes, has been described to produce a range of metabolites that have insecticidal,antibacterial，anticancer activity, and so on. At present, these bacteria have been regardedas an important microbial resource and have huge development potential and goodapplication prospect. In particular, entomopathogenic bacteria can produce a range ofnovel toxin proteins that could be directly applied as protein insecticides or used in thetransgenic plants and microbes. Xenorhabdus budapestensis D43live in symbiosis withentomopathogenic nematode Steinernema ceratophorum, which was isolated fromnortheast China soils by using the insect bait entrapment method. In this study, we directlypurified new intracellular protein fractions from X. budapestensis D43that were screenedby insect bioassay. Subsequently, new insecticidal protein was studied in various aspectsinclude protein identification, gene cloning, bioinformatic analysis, recombinant proteinexpression and purification, and pathogenetic mechanism. The main results of this studycan be summarized as follows:
1. In this report, we purified four protein fractions from the intracellular extract of X.budapestensis D43by preparative native PAGE. However, only one protein exhibitedinsecticidal activity against Galleria mellonella larvae by injection that was designatedHIP57. By injection, HIP57caused G. mellonella larval bodies to blacken and die with anLD50of206.81ng/larva. Especially, when we injected no less than490ng of HIP57perlarva, almost all G. mellonella larvae developed blackened body color within15min anddead after forty-eight hours.
2. HIP57has a molecular weight (MW) of57379.7Da and a theoretical isoelectric point(pI) of4.77. The full-length1647bp sequence of hip57was obtained by sequencing and itssequence has been deposited in GenBank (accession no. JN863588), whereas its deducedamino acid sequence of548amino acids was deposited as AEU10771(GenBank accessionno.). HIP57protein is homologous to GroEL proteins from other bacteria. GroEL has beenaccepted as molecule chaperon, however, our research revealed that HIP57(GroEL)possesses another novel function as an insecticide in X. budapestensis D43.
3. A bioinformatic analysis suggested that signal peptide is absent in HIP57protein, whichis stable and soluble in water. HIP57secondary structure prediction has been performed byfour different methods. At the same time, we predicted the accurate three-dimensional structure of HIP57with high accuracy by homology modeling. By multiple sequencealignment, we found that HIP57contained several specific amino acid residues, which didnot appear in any of the other four GroEL proteins from the family of mutualistic bacteriaat the same positions. A phylogenetic tree was constructed base on the homologues offorty-three GroEL proteins from the family of Enterobacteriaceae. In the phylogenetic tree,X. budapestensis D43were successfully classified into Xenorhabdus, which indicated thatGroEL have potential application in bacterial taxonomy and evolution.
4. The recombinant HIP57, GFP and HIP57-GFP proteins were respectively expressed andpurified from transformed E. coli. Western blotting analysis show that these recombinantproteins could bind specifically to antibodies, respectively. Compared with the case ofoccurring melanism involve darkening of the whole body of wax moth larvae injected withnatural HIP57protein, recombinant HIP57protein did not show similar virulence.However, HIP57-GFP recombinant proteins was enable for the formation of black nodulein the haemocoel of larvae, but not lethal.
5. G. mellonella larvae were injected with HIP57, resulting in extensive immune responsesinclude a significant increase in PO activity, a significant decline in hemocyte populationsand encapsulation response of hemocytes, compared to the controls. Our results illustratepronounced changes in the expression of four genes encoding hemolymph proteins,including ApoLp-III、Serpin、P27K and GST, were observed after HIP57exposure. Weinvestigated the hemolymph proteins that interact with HIP57protein usingco-immunoprecipitation. In vitro two interactions were arylphorin and β-1,3-glucanrecognition protein precursor, which were identified.
In this research we unsuccessfully answer the problem that is if HIP57was secreted byentomopathogenic nematode-bacteria complex during infection host insect. However,HIP57show high lethal potency in the G. mellonella larva bioassay, thereby hypothesizingthat the bacteria could secret HIP57, which may play an important role inentomopathogenic nematode-bacteria complex infecting host insect. Even if the abovehypothesis is not confirmed, HIP57has research significance and application potency as aresult of excellent pathogenicity and novel insecticide mode. The results of my study forPh.D. degree are of greatly importance to elucidate the pathogenetic mechanism of HIP57,which will be useful for appropriate application of HIP57.
引文
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