苏云金杆菌高效杀虫工程菌的构建及其伴胞晶体形成机制研究
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摘要
苏云金芽胞杆菌(Bacillus thuringiensis)简称Bt)作为一种环境友好型生物杀虫剂,对靶标害虫具有专一的杀虫活性,因此被广泛使用于农林业虫害以及蚊媒传播疾病的控制。它的杀虫特性主要来源于在芽胞形成期大量产生的杀虫活性蛋白。尽管在生态环境保护方面具有无可比拟的优势,但Bt生物杀虫剂的使用仍然落后于合成的化学农药。对Bt菌株进行遗传改良,可为发展新型Bt生物杀虫剂产品提供更好的技术与菌种资源。
为获得毒力更高、杀虫谱更广的Bt菌株,进一步扩展Bt杀虫剂在生物防治上的应用,本研究采用分子手段,利用Bt偏爱密码子优化设计了昆虫特异的钙离子通道阻断剂基因ω-ACTX-Hv1a,并进行化学合成,与杀虫晶体蛋白基因crylAc3’-端融合,在Bt中进行共表达,并研究了融合蛋白的晶体形成情况和生物活性功能。在Bt杀虫蛋白基因crylAc的强表达体系调控下,cry1Ac、ω-ACTX-Hv1a和绿色荧光蛋白基因egfp在Bt无晶体突变株Cry-B中得到了有效的融合表达。通过显微镜观察发现,Bt融合基因工程菌Cry-B (1Ac-ACTX-EGFP)的单个菌体中形成了2-3个较小的类似伴胞晶体的包涵体,在菌体裂解后仍然是稳定的。在对Bt工程菌表达产物进行的免疫杂交分析中,全长166kDa的融合蛋白带被清晰的检测到,说明融合基因可以在Bt中进行完整的表达。通过对工程菌发酵产物进行生物活性测定,发现融合蛋白晶体对甜菜夜蛾幼虫的毒力比CrylAc晶体提高了至少5倍,对棉铃虫幼虫也具有很高的毒力且明显抑制了幼虫的生长。这些结果说明,外源杀虫蛋白可以采取与Cry类内毒素C-端融合的方式在Bt中共表达,共同形成伴胞晶体,并至少保留部分杀虫生物活性。本研究为构建具有不同杀虫特性的融合蛋白以及构建高效广谱的Bt工程菌株奠定了重要基础。
目前,研究者们虽然对cry基因的表达调控已经进行了广泛研究,但由于在细菌活体中观察Cry蛋白的聚集过程比较困难,缺乏好的检测手段,因此人们对此类蛋白晶体形成的机制仍然很不清楚。本研究以Bt融合基因工程菌Cry-B(1Ac-ACTX-EGFP)为研究对象,利用激光共聚焦扫描显微镜对芽胞期菌体的蛋白晶体形成过程进行了连续时间段的观察。观察结果显示,融合蛋白开始表达时是散布于母细胞内的,晶体形成是一个由蛋白分子从分散到逐渐聚集的一个过程,并且蛋白的聚集位点存在极性,靠近菌体两端。菌体裂解前,融合蛋白形成的晶体颗粒体积达到最大。本研究构建的Cry蛋白可视化分析系统在细胞水平上为深入研究杀虫蛋白晶体的组装与形成机制提供了非常有用的技术检测手段。
通过对Cry-B (1Ac-ACTX-EGFP)和对照菌株Cry-B(1Ac)裂解前的菌体全蛋白做无标记定量的shot-gun质谱鉴定和蛋白表达差异分析,初步探索了可能参与影响杀虫蛋白晶体形成的调控因子。从Cry-B(1Ac-ACTX-EGFP)菌株和Cry-B (1Ac)菌株中分别鉴定到201和138个非冗余蛋白,其中114个为两菌株共同所有。蛋白功能分类发现两个菌株中功能归于氧化还原类(Oxidation-reduction)的蛋白数量所占比例差异很大,说明菌体内的氧化还原环境可能是造成晶体形成差异较大的原因之一。通过蛋白表达差异比较分析,找到了两菌株中丰度差异较大的共有蛋白和丰度较大的独有蛋白,如共同作用参与蛋白折叠与组装的60kDa伴侣蛋白(GroEL)和10kDa伴侣蛋白(GroES)、参与细胞应激反应的Clp类蛋白酶和催化还原过氧化氢的过氧化氢酶等,均有可能是参与Bt杀虫蛋白晶体形成的调控因子。例如同源性较高的GroEL,已经有研究证实参与调控大肠杆菌外源蛋白包涵体的形成。仍然需要进行基因敲除、加倍互补等分子实验,并结合Cryl蛋白可视化分析系统,来进一步验证这些差异蛋白的功能与晶体形成机制的相关性。本研究提供的信息为探索杀虫蛋白晶体的形成机制,以及合理应用基因工程手段对Bt菌进行遗传改良提供了有价值的技术资料和数据。
Bacillus thuringiensis (Bt) has been used as a biopesticide in agriculture, forestry and mosquito control because of its advantages of specific toxicity against target insects, lack of polluting residues and safety to non-target organisms. The insecticidal properties of this bacterium are due to insecticidal proteins produced during sporulation. Despite these ecological benefits, the use of Bt biopesticides has lagged behind the synthetic chemical pesticides. Genetic improvement of Bt strains, offers a promising means of providing technology and strain resources to develop novel Bt-based bioinsecticide products.
In order to obtain Bt strains with enhanced toxicity and large-spectrum to improve the application of Bt insecticides, we designed and synthesized the DNA sequence of insect-specific VGCCs blockerω-ACTX-Hvla according to the codon preference of the Bt strain, and fused it to the3'-end of insecticidal crystal protein gene cry1Ac. Then we investigated the expression, crystallization, and functional activity of the fusion protein in Bt strain. The fusion gene of cry1Ac,ω-ACTX-Hv1a and enhanced green fluorescent protein gene egfp was expressed in Bt acrystalliferous strain Cry-B under the control of the native gene crylAc expression system. The fusion recombinant Cry-B(1Ac-ACTX-EGFP) generally produced two or three small crystal-like inclusion bodies in each cell. A166kDa full-length fusion protein was identified by immunoblot analysis. This convinced that the fusion gene could be intactly expressed in Bt strain. The bioassayes of lysis of recombinant strain indicated that, virulence of the fusion inclusions was at least fivefold higher than CrylAc crystals toward larvae of Spodoptera exigua. It was also toxic to Helicoverpa armigera larvae and significantly retarded the growth of the larvae. These results demonstrated that a foreign protein could be expressed and accumulate as parasporal inclusions in Bt by C-terminal fusion with the native endotoxin while retaining partial insecticidal activity. The study provides an important foundation for constructing fusion protein with different insecticide activity and recombinant strains with higher toxicity.
Although the expression of cryl genes has been widely studied, the mechanisms of crystal formation and assembly in Bt strains remain unclear, because it is relatively difficult to study the assembly and aggregation of Cry proteins in individual cells in vivo. In this work, we used the engineering strain Cry-B(1Ac-ACTX-EGFP) as the object, observed crystallization of the fusion protein in samples collected at consequent time point with confocal laser scanning microscopy. The result showed that, at the beginning of expression, the fusion protein was distributed in the mother cell. The formation of crystal is a process that can be compared to protein molecule polymerization. Surprisingly, the fusion protein crystals showed polarity and were located near two ends of the strain. Before cell lysis, the crystals grew to full size. This reporter system may be a useful tool to study the mechanisms of parasporal crystal formation and assembly.
Then, a comparative proteomic analysis was performed on the Bt engineering strain Cry-B (1Ac-ACTX-EGFP) and Cry-B (1Ac) before cell lysis using label-free quantitative shotgun proteomics to investigate the underlying regulators participating in formation of insecticidal protein crystals. In total,201proteins from Cry-B (1Ac-ACTX-EGFP) and138proteins from Cry-B (1Ac) were unambiguously identified by liquid chromatography-tandem mass spectrometry analysis, in which,114proteins were shared by the two strains. The result of protein functional categorization demonstrated that, the proportion (in percentage) of identified proteins related to oxidation-reduction showed great changes between the two strains. This indicated that the different oxidation-reduction environment inside the cell may be one of reasons caused crystal difference between the two strains. We found the proteins with notable difference in expression level between the two strains and the proteins with a high emPAI value only identified in one strain, such as60kDa chaperonin (GroEL) and10kDa chaperonin (GroES) involved in preventing protein's misfolding and promotes the refolding and assembly; Clp protease involved in cell's response to stress; catalase that catalyzing reduction of hydrogen peroxide and so on. These proteins may be the regulators participating in crystals' formation. Certainly we still need do more molecular experiments and combine the GFP reporter system to test and verify the relationship between the function of these proteins and the mechanisms of crystal formation. This work provided valuable technologic information and data for exploring the mechanisms of crystal formation and further improving Bt strains by rational application of genetic engineering.
为获得毒力更高、杀虫谱更广的Bt菌株,进一步扩展Bt杀虫剂在生物防治上的应用,本研究采用分子手段,利用Bt偏爱密码子优化设计了昆虫特异的钙离子通道阻断剂基因ω-ACTX-Hv1a,并进行化学合成,与杀虫晶体蛋白基因crylAc3’-端融合,在Bt中进行共表达,并研究了融合蛋白的晶体形成情况和生物活性功能。在Bt杀虫蛋白基因crylAc的强表达体系调控下,cry1Ac、ω-ACTX-Hv1a和绿色荧光蛋白基因egfp在Bt无晶体突变株Cry-B中得到了有效的融合表达。通过显微镜观察发现,Bt融合基因工程菌Cry-B (1Ac-ACTX-EGFP)的单个菌体中形成了2-3个较小的类似伴胞晶体的包涵体,在菌体裂解后仍然是稳定的。在对Bt工程菌表达产物进行的免疫杂交分析中,全长166kDa的融合蛋白带被清晰的检测到,说明融合基因可以在Bt中进行完整的表达。通过对工程菌发酵产物进行生物活性测定,发现融合蛋白晶体对甜菜夜蛾幼虫的毒力比CrylAc晶体提高了至少5倍,对棉铃虫幼虫也具有很高的毒力且明显抑制了幼虫的生长。这些结果说明,外源杀虫蛋白可以采取与Cry类内毒素C-端融合的方式在Bt中共表达,共同形成伴胞晶体,并至少保留部分杀虫生物活性。本研究为构建具有不同杀虫特性的融合蛋白以及构建高效广谱的Bt工程菌株奠定了重要基础。
目前,研究者们虽然对cry基因的表达调控已经进行了广泛研究,但由于在细菌活体中观察Cry蛋白的聚集过程比较困难,缺乏好的检测手段,因此人们对此类蛋白晶体形成的机制仍然很不清楚。本研究以Bt融合基因工程菌Cry-B(1Ac-ACTX-EGFP)为研究对象,利用激光共聚焦扫描显微镜对芽胞期菌体的蛋白晶体形成过程进行了连续时间段的观察。观察结果显示,融合蛋白开始表达时是散布于母细胞内的,晶体形成是一个由蛋白分子从分散到逐渐聚集的一个过程,并且蛋白的聚集位点存在极性,靠近菌体两端。菌体裂解前,融合蛋白形成的晶体颗粒体积达到最大。本研究构建的Cry蛋白可视化分析系统在细胞水平上为深入研究杀虫蛋白晶体的组装与形成机制提供了非常有用的技术检测手段。
通过对Cry-B (1Ac-ACTX-EGFP)和对照菌株Cry-B(1Ac)裂解前的菌体全蛋白做无标记定量的shot-gun质谱鉴定和蛋白表达差异分析,初步探索了可能参与影响杀虫蛋白晶体形成的调控因子。从Cry-B(1Ac-ACTX-EGFP)菌株和Cry-B (1Ac)菌株中分别鉴定到201和138个非冗余蛋白,其中114个为两菌株共同所有。蛋白功能分类发现两个菌株中功能归于氧化还原类(Oxidation-reduction)的蛋白数量所占比例差异很大,说明菌体内的氧化还原环境可能是造成晶体形成差异较大的原因之一。通过蛋白表达差异比较分析,找到了两菌株中丰度差异较大的共有蛋白和丰度较大的独有蛋白,如共同作用参与蛋白折叠与组装的60kDa伴侣蛋白(GroEL)和10kDa伴侣蛋白(GroES)、参与细胞应激反应的Clp类蛋白酶和催化还原过氧化氢的过氧化氢酶等,均有可能是参与Bt杀虫蛋白晶体形成的调控因子。例如同源性较高的GroEL,已经有研究证实参与调控大肠杆菌外源蛋白包涵体的形成。仍然需要进行基因敲除、加倍互补等分子实验,并结合Cryl蛋白可视化分析系统,来进一步验证这些差异蛋白的功能与晶体形成机制的相关性。本研究提供的信息为探索杀虫蛋白晶体的形成机制,以及合理应用基因工程手段对Bt菌进行遗传改良提供了有价值的技术资料和数据。
Bacillus thuringiensis (Bt) has been used as a biopesticide in agriculture, forestry and mosquito control because of its advantages of specific toxicity against target insects, lack of polluting residues and safety to non-target organisms. The insecticidal properties of this bacterium are due to insecticidal proteins produced during sporulation. Despite these ecological benefits, the use of Bt biopesticides has lagged behind the synthetic chemical pesticides. Genetic improvement of Bt strains, offers a promising means of providing technology and strain resources to develop novel Bt-based bioinsecticide products.
In order to obtain Bt strains with enhanced toxicity and large-spectrum to improve the application of Bt insecticides, we designed and synthesized the DNA sequence of insect-specific VGCCs blockerω-ACTX-Hvla according to the codon preference of the Bt strain, and fused it to the3'-end of insecticidal crystal protein gene cry1Ac. Then we investigated the expression, crystallization, and functional activity of the fusion protein in Bt strain. The fusion gene of cry1Ac,ω-ACTX-Hv1a and enhanced green fluorescent protein gene egfp was expressed in Bt acrystalliferous strain Cry-B under the control of the native gene crylAc expression system. The fusion recombinant Cry-B(1Ac-ACTX-EGFP) generally produced two or three small crystal-like inclusion bodies in each cell. A166kDa full-length fusion protein was identified by immunoblot analysis. This convinced that the fusion gene could be intactly expressed in Bt strain. The bioassayes of lysis of recombinant strain indicated that, virulence of the fusion inclusions was at least fivefold higher than CrylAc crystals toward larvae of Spodoptera exigua. It was also toxic to Helicoverpa armigera larvae and significantly retarded the growth of the larvae. These results demonstrated that a foreign protein could be expressed and accumulate as parasporal inclusions in Bt by C-terminal fusion with the native endotoxin while retaining partial insecticidal activity. The study provides an important foundation for constructing fusion protein with different insecticide activity and recombinant strains with higher toxicity.
Although the expression of cryl genes has been widely studied, the mechanisms of crystal formation and assembly in Bt strains remain unclear, because it is relatively difficult to study the assembly and aggregation of Cry proteins in individual cells in vivo. In this work, we used the engineering strain Cry-B(1Ac-ACTX-EGFP) as the object, observed crystallization of the fusion protein in samples collected at consequent time point with confocal laser scanning microscopy. The result showed that, at the beginning of expression, the fusion protein was distributed in the mother cell. The formation of crystal is a process that can be compared to protein molecule polymerization. Surprisingly, the fusion protein crystals showed polarity and were located near two ends of the strain. Before cell lysis, the crystals grew to full size. This reporter system may be a useful tool to study the mechanisms of parasporal crystal formation and assembly.
Then, a comparative proteomic analysis was performed on the Bt engineering strain Cry-B (1Ac-ACTX-EGFP) and Cry-B (1Ac) before cell lysis using label-free quantitative shotgun proteomics to investigate the underlying regulators participating in formation of insecticidal protein crystals. In total,201proteins from Cry-B (1Ac-ACTX-EGFP) and138proteins from Cry-B (1Ac) were unambiguously identified by liquid chromatography-tandem mass spectrometry analysis, in which,114proteins were shared by the two strains. The result of protein functional categorization demonstrated that, the proportion (in percentage) of identified proteins related to oxidation-reduction showed great changes between the two strains. This indicated that the different oxidation-reduction environment inside the cell may be one of reasons caused crystal difference between the two strains. We found the proteins with notable difference in expression level between the two strains and the proteins with a high emPAI value only identified in one strain, such as60kDa chaperonin (GroEL) and10kDa chaperonin (GroES) involved in preventing protein's misfolding and promotes the refolding and assembly; Clp protease involved in cell's response to stress; catalase that catalyzing reduction of hydrogen peroxide and so on. These proteins may be the regulators participating in crystals' formation. Certainly we still need do more molecular experiments and combine the GFP reporter system to test and verify the relationship between the function of these proteins and the mechanisms of crystal formation. This work provided valuable technologic information and data for exploring the mechanisms of crystal formation and further improving Bt strains by rational application of genetic engineering.
引文
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