苏云金杆菌4.0718新菌株不同生长时期的蛋白质组研究
摘要
本研究提取了苏云金芽胞杆菌(Bacillus thuringiensis,Bt)库斯塔克亚种4.0718新菌株营养生长中期(T_1)、芽胞形成前期(T_2)和芽胞后期(T_3)三个时期的细胞总蛋白质,采用1DE-MS/MS,2DE-MS/MS和“shotgun”技术分离和鉴定蛋白质。比较了不同生长时期细胞总蛋白质表达谱之间的差异,并分析了包括杀虫晶体蛋白质(Insecticidal Crystal Proteins,ICPs)在内的功能蛋白质的表达时空特性,获得了与晶体和芽胞形成过程相关的重要蛋白质信息。
利用Melanie6.0软件分析2DE图谱,发现在T_1、T_2和T_3时期的蛋白质点数目分别为346±18,299±23和343±11。以T_2时期的2DE胶为参照,T_1和T_3时期的2DE胶与之对比,匹配率分别为70%和60%,反应了蛋白质在不同生长时期的表达差异性。切取蛋白质点并进行MALDI-TOF/TOF MS分析和数据库搜寻,鉴定差异表达的蛋白质点。结果发现,在鉴定到的差异蛋白质点中,T_1时期特异性表达的有蛋白酶VCA0223和转酮醇酶等9个蛋白质点;T_2时期特异性表达的3个蛋白质点分别为支链氨基酸转氨酶、嘌呤核苷磷酸化酶和假想蛋白BC1708;T_3时期特异性表达20个蛋白质点,包括bacillolysin、ORF1和SpoIVA等。与T_2时期比较,T_1时期无表达,T_3时期表达上调的蛋白质点为Cry1A(c)3;下调的蛋白质点6个,分别为寡肽接合蛋白OppA、肽酶T和有机耐受蛋白等;无显著变化的蛋白质点为NAD(+)合成酶和果糖二磷酸醛缩酶。与T_1时期比较,T_3时期无表达,T_2时期表达上调的2个蛋白质点分别为免疫因子A(Immune Inhibitor A,InhA)和InhA precursor;下调的4个蛋白质点包括氨甲基转移酶和转录抑制因子CodY等;无显著变化的2个蛋白质点为异柠檬酸脱氢酶和3-ketoacyl-酰基载体蛋白还原酶。在三个时期均有表达,但相对T_1时期,1-吡咯啉-5-羧酸脱氢酶和苹果酸脱氢酶等5个蛋白质点在T_2时期表达显著下调,苏氨酸脱氢酶在T_2时期表达显著上调,腺苷琥珀酸裂解酶在T_3时期表达显著下调。
采用1DE-MS/MS和shotgun技术分离和鉴定到了2DE-MS/MS未分离得到的重要功能蛋白质Cry2Aa和ORF2,发现Cry2Aa在T_2时期开始形成,ORF2只在T_3时期表达,这是对2DE数据的一个重要补充。
对差异蛋白质的表达特征进行综合分析,发现T_1时期表达的蛋白质主要为物质代谢和能量代谢过程中的重要酶类,与其它芽胞杆菌生长期细胞的表达谱相似。T_2时期部分柠檬酸循环酶类蛋白质的表达下调,说明细胞代谢速度开始下降,细胞营养受限,为芽胞形成提供了重要条件,芽胞开始形成,ICPs起始高效表达。T_3时期代谢相关蛋白质的表达量进一步降低,ICPs表达量最高,并形成晶体结构,有利于细胞逆境生存的有机溶剂耐受蛋白和PspA的表达在维护细胞完整和晶体形成中发挥作用。这是首次利用蛋白质组技术研究Bt细胞生理活性物质的变化情况,为深入研究Bt细胞的生理代谢过程、各生物活性因子的功能及探讨Bt的杀虫机制奠定了重要基础,也为拓宽Bt杀虫谱,增强杀虫效果提供了科学依据。同时通过对CotJC、ORF2和SpoIVh等蛋白质的表达特征和功能的分析,获得了与晶体和芽胞形成过程相关的重要信息,为研究晶体蛋白的稳定表达及如何构建稳定抗降解的晶体蛋白质结构奠定了基础。
The proteins of whole cells of Bacillus thuringiensis strain 4.0718 at three phases-middle vegetative phase(T_1), early sporulation phase(T_2)and late sporulation phase(T_3) were extracted,followed with separation and identification by 1DE-MS/MS,2DE-MS/MS and proteomic shotgun technologies. The proteomic profiles were compared,and characteristics of functional proteins including insecticidal crystal proteins (ICPs)were analyzed to get information about the construction of crystals and spores.
In this study,2DE maps were analyzed using Melanie6.0 software.Analysis results showed that the number of spots in 2DE gels of protein samples of cells at T_1,T_2 and T_3 was 346±18,299±23 and 343±11,respectively.The matching ratio of 2DE gels of protein samples of T_1 and T_3,to 2DE gel of the protein sample of T_2,which was set as reference gel,was 70%and 60%, respecticely.The comparison suggested the characteristics of protein expression at different phases.Protein spots were cut out and analyzed by MALDI-TOF/TOF MS and database search,to identify the proteins with expression change at different phases.The results showed that 9 specifically expressed proteins were identified at T_1,including protease VCA0223 and transketolase;3 at T_2,such as purine nucleoside phosphorylase, hypothetical protein BC1708 and branched-chain amino acid aminotransferase;and 20 at T-3 including bacillolysin,ORF1 and SpoIVA and so on.9 proteins began their expression at T_2,in which,CryiA(c)3 was upregulated at T_3;6 proteins including OppA,peptidase T and organic tolerance protein were downregulated,and 2 proteins-NAD(+)synthase and fructose-bisphosphate aldolase were not regulated significant at T_3.Compared with the expression at T_1,immune inhibitor A (InhA)and InhA precursor were upregulated at T_2,while 4 proteins including CodY protein and aminomethyltransferase were downregulated,and isocitrate dehydrogenase and 3-ketoacyl-(acyl-carrier-protein)reductase were not regulated at T_2.The 8 proteins described above were not found at T_3.The proteins mentioned below were all identified at three phases.Compared with the expression at T_1,5 proteins were downregulated at T_2,for example,1-pyrroline-5-carboxylate dehydrogenase and malate dehydrogenase.And adenylosuccinate synthetase was downregulated at T_3,while threonine synthase was upregulated at T_2.
By use of IDE-MS/MS and shotgun technologies,Cry2Aa and ORF2 with important function were identified,as important supplementary to 2DE data.Cry2Aa began to express at T_2,while ORF2 specifically expressed at T_3.
Analysis on expression characteristics of differentially expressed proteins showed that the main proteins were enzymes participating in metabolism pathways at T_1,similar to expression profile of growing cells of other Bacillus.Parts of proteins involved in citric acid cycle were downregulated at T_2,suggesting nutrition limited state of cells,which provided an important prerequisite for the formation of spores. At the same time,ICPs started to express efficiently and spores began to form.The expression amount of proteins related to metabolism further reduced at T_3;ICPs reached the highest expression,and formed into crystal structure,The expression of organic tolerance protein and PspA protein at T_3 could maintain the integrity of cells and the formation of crystals. This is the first use of proteomic technologies to research life process of B.thuringiensis and detect bioactive factors.It laid an important foundation for the study of its physiological process,the function of bioactive factors and the insecticide mechanisms in depth,and provided scientific basis for studying how to broaden the insecticide spectrum,and to enhance the killing effect of B.thuringiensis.Meanwhile,through analysis on expression change and function of proteins such as CotJC,ORF2 and SpoIVA,important informations about the formation of crystals and spores were got,providing a basis for further exploring the stable expression of ICPs and the formation of crystals resistant to degredation.
利用Melanie6.0软件分析2DE图谱,发现在T_1、T_2和T_3时期的蛋白质点数目分别为346±18,299±23和343±11。以T_2时期的2DE胶为参照,T_1和T_3时期的2DE胶与之对比,匹配率分别为70%和60%,反应了蛋白质在不同生长时期的表达差异性。切取蛋白质点并进行MALDI-TOF/TOF MS分析和数据库搜寻,鉴定差异表达的蛋白质点。结果发现,在鉴定到的差异蛋白质点中,T_1时期特异性表达的有蛋白酶VCA0223和转酮醇酶等9个蛋白质点;T_2时期特异性表达的3个蛋白质点分别为支链氨基酸转氨酶、嘌呤核苷磷酸化酶和假想蛋白BC1708;T_3时期特异性表达20个蛋白质点,包括bacillolysin、ORF1和SpoIVA等。与T_2时期比较,T_1时期无表达,T_3时期表达上调的蛋白质点为Cry1A(c)3;下调的蛋白质点6个,分别为寡肽接合蛋白OppA、肽酶T和有机耐受蛋白等;无显著变化的蛋白质点为NAD(+)合成酶和果糖二磷酸醛缩酶。与T_1时期比较,T_3时期无表达,T_2时期表达上调的2个蛋白质点分别为免疫因子A(Immune Inhibitor A,InhA)和InhA precursor;下调的4个蛋白质点包括氨甲基转移酶和转录抑制因子CodY等;无显著变化的2个蛋白质点为异柠檬酸脱氢酶和3-ketoacyl-酰基载体蛋白还原酶。在三个时期均有表达,但相对T_1时期,1-吡咯啉-5-羧酸脱氢酶和苹果酸脱氢酶等5个蛋白质点在T_2时期表达显著下调,苏氨酸脱氢酶在T_2时期表达显著上调,腺苷琥珀酸裂解酶在T_3时期表达显著下调。
采用1DE-MS/MS和shotgun技术分离和鉴定到了2DE-MS/MS未分离得到的重要功能蛋白质Cry2Aa和ORF2,发现Cry2Aa在T_2时期开始形成,ORF2只在T_3时期表达,这是对2DE数据的一个重要补充。
对差异蛋白质的表达特征进行综合分析,发现T_1时期表达的蛋白质主要为物质代谢和能量代谢过程中的重要酶类,与其它芽胞杆菌生长期细胞的表达谱相似。T_2时期部分柠檬酸循环酶类蛋白质的表达下调,说明细胞代谢速度开始下降,细胞营养受限,为芽胞形成提供了重要条件,芽胞开始形成,ICPs起始高效表达。T_3时期代谢相关蛋白质的表达量进一步降低,ICPs表达量最高,并形成晶体结构,有利于细胞逆境生存的有机溶剂耐受蛋白和PspA的表达在维护细胞完整和晶体形成中发挥作用。这是首次利用蛋白质组技术研究Bt细胞生理活性物质的变化情况,为深入研究Bt细胞的生理代谢过程、各生物活性因子的功能及探讨Bt的杀虫机制奠定了重要基础,也为拓宽Bt杀虫谱,增强杀虫效果提供了科学依据。同时通过对CotJC、ORF2和SpoIVh等蛋白质的表达特征和功能的分析,获得了与晶体和芽胞形成过程相关的重要信息,为研究晶体蛋白的稳定表达及如何构建稳定抗降解的晶体蛋白质结构奠定了基础。
The proteins of whole cells of Bacillus thuringiensis strain 4.0718 at three phases-middle vegetative phase(T_1), early sporulation phase(T_2)and late sporulation phase(T_3) were extracted,followed with separation and identification by 1DE-MS/MS,2DE-MS/MS and proteomic shotgun technologies. The proteomic profiles were compared,and characteristics of functional proteins including insecticidal crystal proteins (ICPs)were analyzed to get information about the construction of crystals and spores.
In this study,2DE maps were analyzed using Melanie6.0 software.Analysis results showed that the number of spots in 2DE gels of protein samples of cells at T_1,T_2 and T_3 was 346±18,299±23 and 343±11,respectively.The matching ratio of 2DE gels of protein samples of T_1 and T_3,to 2DE gel of the protein sample of T_2,which was set as reference gel,was 70%and 60%, respecticely.The comparison suggested the characteristics of protein expression at different phases.Protein spots were cut out and analyzed by MALDI-TOF/TOF MS and database search,to identify the proteins with expression change at different phases.The results showed that 9 specifically expressed proteins were identified at T_1,including protease VCA0223 and transketolase;3 at T_2,such as purine nucleoside phosphorylase, hypothetical protein BC1708 and branched-chain amino acid aminotransferase;and 20 at T-3 including bacillolysin,ORF1 and SpoIVA and so on.9 proteins began their expression at T_2,in which,CryiA(c)3 was upregulated at T_3;6 proteins including OppA,peptidase T and organic tolerance protein were downregulated,and 2 proteins-NAD(+)synthase and fructose-bisphosphate aldolase were not regulated significant at T_3.Compared with the expression at T_1,immune inhibitor A (InhA)and InhA precursor were upregulated at T_2,while 4 proteins including CodY protein and aminomethyltransferase were downregulated,and isocitrate dehydrogenase and 3-ketoacyl-(acyl-carrier-protein)reductase were not regulated at T_2.The 8 proteins described above were not found at T_3.The proteins mentioned below were all identified at three phases.Compared with the expression at T_1,5 proteins were downregulated at T_2,for example,1-pyrroline-5-carboxylate dehydrogenase and malate dehydrogenase.And adenylosuccinate synthetase was downregulated at T_3,while threonine synthase was upregulated at T_2.
By use of IDE-MS/MS and shotgun technologies,Cry2Aa and ORF2 with important function were identified,as important supplementary to 2DE data.Cry2Aa began to express at T_2,while ORF2 specifically expressed at T_3.
Analysis on expression characteristics of differentially expressed proteins showed that the main proteins were enzymes participating in metabolism pathways at T_1,similar to expression profile of growing cells of other Bacillus.Parts of proteins involved in citric acid cycle were downregulated at T_2,suggesting nutrition limited state of cells,which provided an important prerequisite for the formation of spores. At the same time,ICPs started to express efficiently and spores began to form.The expression amount of proteins related to metabolism further reduced at T_3;ICPs reached the highest expression,and formed into crystal structure,The expression of organic tolerance protein and PspA protein at T_3 could maintain the integrity of cells and the formation of crystals. This is the first use of proteomic technologies to research life process of B.thuringiensis and detect bioactive factors.It laid an important foundation for the study of its physiological process,the function of bioactive factors and the insecticide mechanisms in depth,and provided scientific basis for studying how to broaden the insecticide spectrum,and to enhance the killing effect of B.thuringiensis.Meanwhile,through analysis on expression change and function of proteins such as CotJC,ORF2 and SpoIVA,important informations about the formation of crystals and spores were got,providing a basis for further exploring the stable expression of ICPs and the formation of crystals resistant to degredation.
引文
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