苏云金芽孢杆菌Cry蛋白分离纯化及其增效研究
摘要
鳞翅目害虫棉铃虫(Helicoverpa armigera)、甜菜夜蛾(Spodoptera exigua)、玉米螟(Ostrina furnacalis)、大豆食心虫(Leguminivora glycintvorella)是农业上的重要害虫，近年来棉铃虫和甜菜夜蛾在田间对化学农药的抗药性上升较快，而玉米螟和大豆食心虫属钻蛀型害虫，使用传统的方法已难以达到良好的防治效果。用苏云金芽孢杆菌杀虫晶体蛋白(ICP)来防治棉铃虫、甜菜夜蛾、玉米螟已有许多报道，但主要都是选用Cry1Ac、Cry1Ab灿作为杀虫蛋白，种类单一，害虫易产生抗性；大豆食心虫是我国北方发生较为严重的害虫，它的为害，直接影响大豆的品质和产量，国内外尚未见对此类害虫有活性的Bt的正式报道，因此选用Bt来防治大豆食心虫将具有很大的市场潜力。本论文针对这些重要的害虫从Bt资源中筛选新的高效Cry蛋白及不同的Cry蛋白组合，对于构建高毒力、广谱、延缓害虫抗药性的新一带工程菌和转基因植物都将具有重要的理论和实践意义。
本研究在分离纯化Bt Cry蛋白的基础上，研究了Cry1Ac、Cry1Ca7、Cry1Cb2、Cry1Ia8、Cry1Ie1和Cry2Ab4六种蛋白酶解情况和杀虫活性分析，包括各原毒素、毒素蛋白单独及两两正交组合，对棉铃虫、甜菜夜蛾、玉米螟、大豆食心虫的毒力，并寻找到对上述害虫高毒力的Bt Cry蛋白和协同增效蛋白组合。
1．Bt Cry蛋白特性的研究
分析比较了在E.coli表达的Cry1Ca7、Cry1Cb2、Cry1Ia8、Cry1Ie1、Cry2Ab4蛋白和标准菌株Cry1Ac(HD-73)蛋白的溶解性、trypsin的消化作用及各自受体菌的生长速率。
通过SDS-PAGE分析发现Cry1Ca7、Cry1Cb2、Cry1Ia8、Cry1Ie1、Cry2Ab4和Cry1Ac原毒素及毒素在50mM Na_2CO_3(pH=10.0)中的溶解度大于在20mM乙醇胺(pH=10.0)中的溶解度。Cry蛋白溶解度的高低直接影响分离纯化的得率，以及最终的数据分析，从而影响Cry蛋白的毒力。
Trypsin是昆虫体内存在的一种蛋白水解酶，它对Cry蛋白的作用结果关系到Cry蛋白毒性的发挥。本论文研究了Cry1Ca7、Cry1Cb2、Cry1Ia8、Cry1Iel、Cry2Ab4和Cry1Ac在50mM Na_2CO_3和20mM乙醇胺(pH=10.0)溶液中被trypsin消化的情况，结果表明只有在适当的浓度下Cry蛋白才会降解成毒素，若trypsin浓度过高，Cry蛋白有可能完全降解。在trypsin：Cry蛋白=1:50时，Cry1Ca7、Cry1Ia8经trypsin消化得到约55kDa大小的多肽。Cry1Cb2，Cry1Iel、Cry2Ab4蛋白酶解得到57kDa的片段，其中Cry1Ia8和Cry1Iel蛋白的trypsin酶解分析研究国内外尚无正式报道。而Cry1Ac经trypsin处理后约
乐北衣业大学硕土论文 苏云金芽抱杆菌Cry蛋白分离纯化及其增效研究
为6皿Da的多肽，与前人报追相符。这几种蛋白在50血MNZ＊0。和20mM乙醇胺
（pH—10、0）溶液中消化的结果相同，只是在20mM乙醇胺（pH＝100）＄液中梢化后的
得率相对低一些。
在LB液体培养基中，这儿株菌株的生长速率均较受体菌BLZI（DE3）慢，可能由于了
外源基因的大量表达影响了受体菌的正常生长发育。在加入诱导剂 IPTG Zhrs后，这几株
菌株先后进入对数生长期，其顺序为 CylCa7、CIy1CbZ、Cryllel、Cryllas，最迟进入
对数生长期的是 CryZAb4，并分别于 12、16、16、16、20hrs进人稳定生长期。而 Bt菌
株HD．刀是在生长4小时后进入对数生长期，！2小时后进入稳定生长期。
2·＊【Cr）、蛋白分离纯化的研究
研究了 Bt菌株和在Ecoli中 Bt Cry蛋白的提取时机，先后分离纯化了印·ICa7。
Crymm、＊nlla巳＊nnel、吐2汕4蛋白和标准菌株＊ilAc（＊D一刀）蛋白，研究
表明在 ECCll中表达的蛋白均在培养 lershrs含量达到最高，CylAC晶体蛋白则是在培养
28～32hrs达到最高c将这几种蛋白经 FPLC纯化并进行了活性分析表明 CylAc在 sedml
处的洗脱峰活性较高，而在CO，基因ECOli中表达产物则是在IDellnd处有较高的活性。
3、Bt Cr）蛋白对棉铃虫幼虫毒性的研究
将上述 6种 Cy蛋白对棉铃虫进行初筛和两两正交组合的复筛．结果表明 CylAc和
CnZAb4对棉铃虫的毒性较强，LC。。分别为刀．sug／thl和415ghl；Cy1Ac＋Cn·ZAb4组
合的LC。；；为48．7twml，两者的共毒系数（C。TC）为121，具有一定的增效作用。其余
毒素对棉铃虫毒力的强弱依次是 Cryllel、Cryllas、CylCa7、CrylCbZ，这几种蛋白的
组合对棉铃虫没有增效作用。
4·Bt Cr3蛋白对玉米螟幼虫毒性的研究
将上述 6种 Cy蛋白对玉米螟进行初筛和两两正交组合的复筛，结果表明q、IAC。
Cn，Ilas。Cn，Ilel对玉米螟有很强的活性，将这三种蛋白进行正交组合对玉米螟进行生测，
发现k，IAc与C口llel、Cfl，ilas分别有明显增效作用、相加作用，用校正死亡率进行
SAS分析 C。IAc、C。·ilas、Cn，Ilel、CrvlAc＋Cryllel、CrylAc＋Cryllas的 LC。。分别
为：0，134、0500、0二23、0046、of98wi。CylAc＋Cry·Ilel和 CrvlAC＋Cryllas蛋白组
合有显著的增效作用，CTC．分别为 409和 106，在 LC。、LC。。水平 CylAC的毒力最
高。这些研究结果说明了CrylAc、Cryrllel、Cry，IAdellel以及CrvlAc＋Cryllas蛋白
组合对玉米螟有很强的杀虫活性，它们完?
Lepidoptera pests, such as cotton boll worm, beet army worm, and com borer worm, were very harmful to many crops and vegetables. Recently, resistance of cotton boll worm and beet army worm for chemicals was increasing quickly in the field, and it was very hard to control some pest by traditional methods. Leguminivora glycinivorella was the most important pest for soybean in north of China. But no formal report involved in the biocontrol using Bt
Bt, the best microbial insecticide, has been reported for control of cotton boll worm, beet worm, but the narrow choice of cry genes, for instance only crylAb or cry1 Ac, may induce the pest resistance.
In this study , different Cry protein expressed in E.coli. and the combinations between Cry proteins were screened against mentioned pests. It was very significant for construction of novel genetically engineered bacteria and transgenic plant for insect pest biocontrol.
1. Biological characteristics of Bt Cry protein
The solubility, digestion of Cry protein with trypsin and growth speed of E.coli. strain were researched respectively.
The solubility of CrylCa7, CrylCb2, Crylla8, CrylIe1, Cry2Ab4 proteins, expressed in E.coli. strains, and CrylAc from HD-73 strain were demonstrated that they were more soluble in Na2CO3 than in Ethanoloanmine by SDS-PAGE analysis.
Trypsin, the hydrdytic enzyme of insects, was crucial for digestion and toxicity of Cry protein. After reaction 6hrs, all the Cry protein were digested, and 55kDa proteins were obtain from CrylCa7 and Crylla8, 57kDa fragment were from CrylCb2, Cryllel and Cry2Ab4, but the 60kDa, peptide was maintained from CrylAc. If the concentration and reaction time were much more , all the Cry protein were degraded completely.
It was the first report of the digestion of Cryllel and CrylIa8 in the LB broth, the growth speed of all the E.coli. strains harboring the recombinant plasmids inserted cry gene, were slower than their host BL21. The over expression of cry gene maybe affect on the growth and development of host cell.
2. Purification of Bt Cry proteins
Expression time of cry protein in Ecoli strain was investigated. After induction by IPTG, 4~6hrs, the accumulation of Cry protein was the most, but prodution of CrylAc from HD-73 strain was the most after 28~32hrs. The result of FPLC showed that all of protoxins and toxins activated by tryspin could be well purified using elutin buffer at pH9.5
3. Bioassay of Cry protein against cotton boll worm larvae
The bioassay result demonstrated that Cry 1 Ac and Cry2Ab4were higher toxicity than other four Cry proteins, and LC50 was 31.5ug/ml and 415ug/ml respectively. Furthermore, the co-toxicity coefficient of Cry lac and Cry2Ab4 combination was 121% according to the LC50 48.7ug/ml of protein combination. It was means that Cry 1 Ac and Cry2Ab4, but other four Cry toxin, could synergize each other.
4. Bioassay of Cry protein against corn borer worm larvae
The result of bioassay indicated that Cry1Ac, Crylla8 and CrylIe1 were high toxicity, and the combination between the three protein, illustrated that CrylIe1+CrylAc combination were synergistic against corn borer worm larvae. LC50 of Cry1Ac, CrylIa8, CrylIe1, CrylAc+CrylIe1 and CrylAc+CrylIa8 was 0.134, 0.500, 0.223, 0.046, and 0.198ug/g respectively. Cotoxicity coefficients of CrylIe1+CrylAc and Crylla8+CrylAc were 409 and 106 separately. Cry1Ac was the highest among the Cry protein on the level of LC90 and LC95. Those bioassay results showed that CrylAc+CrylIe1 combination could be used for construction of engineered bacteria and transgenic plant, and meanwhile Cry1Ac as an enhancer protein could be combinated with other Cry protein for Insecticidal research.
5. Bioassay of Cry protein against beet army worm larvae
The larvae could be inhibited by all the Cry protein, and Cry1Ac and CrylCa7 combination was more toxic.
6. Bioassay of Cry protein against Leguminivora glycinivorella larvae
The result of bioassay of Cry protein to Leguminivora glycinivorella larvae, by two time select
本研究在分离纯化Bt Cry蛋白的基础上，研究了Cry1Ac、Cry1Ca7、Cry1Cb2、Cry1Ia8、Cry1Ie1和Cry2Ab4六种蛋白酶解情况和杀虫活性分析，包括各原毒素、毒素蛋白单独及两两正交组合，对棉铃虫、甜菜夜蛾、玉米螟、大豆食心虫的毒力，并寻找到对上述害虫高毒力的Bt Cry蛋白和协同增效蛋白组合。
1．Bt Cry蛋白特性的研究
分析比较了在E.coli表达的Cry1Ca7、Cry1Cb2、Cry1Ia8、Cry1Ie1、Cry2Ab4蛋白和标准菌株Cry1Ac(HD-73)蛋白的溶解性、trypsin的消化作用及各自受体菌的生长速率。
通过SDS-PAGE分析发现Cry1Ca7、Cry1Cb2、Cry1Ia8、Cry1Ie1、Cry2Ab4和Cry1Ac原毒素及毒素在50mM Na_2CO_3(pH=10.0)中的溶解度大于在20mM乙醇胺(pH=10.0)中的溶解度。Cry蛋白溶解度的高低直接影响分离纯化的得率，以及最终的数据分析，从而影响Cry蛋白的毒力。
Trypsin是昆虫体内存在的一种蛋白水解酶，它对Cry蛋白的作用结果关系到Cry蛋白毒性的发挥。本论文研究了Cry1Ca7、Cry1Cb2、Cry1Ia8、Cry1Iel、Cry2Ab4和Cry1Ac在50mM Na_2CO_3和20mM乙醇胺(pH=10.0)溶液中被trypsin消化的情况，结果表明只有在适当的浓度下Cry蛋白才会降解成毒素，若trypsin浓度过高，Cry蛋白有可能完全降解。在trypsin：Cry蛋白=1:50时，Cry1Ca7、Cry1Ia8经trypsin消化得到约55kDa大小的多肽。Cry1Cb2，Cry1Iel、Cry2Ab4蛋白酶解得到57kDa的片段，其中Cry1Ia8和Cry1Iel蛋白的trypsin酶解分析研究国内外尚无正式报道。而Cry1Ac经trypsin处理后约
乐北衣业大学硕土论文 苏云金芽抱杆菌Cry蛋白分离纯化及其增效研究
为6皿Da的多肽，与前人报追相符。这几种蛋白在50血MNZ＊0。和20mM乙醇胺
（pH—10、0）溶液中消化的结果相同，只是在20mM乙醇胺（pH＝100）＄液中梢化后的
得率相对低一些。
在LB液体培养基中，这儿株菌株的生长速率均较受体菌BLZI（DE3）慢，可能由于了
外源基因的大量表达影响了受体菌的正常生长发育。在加入诱导剂 IPTG Zhrs后，这几株
菌株先后进入对数生长期，其顺序为 CylCa7、CIy1CbZ、Cryllel、Cryllas，最迟进入
对数生长期的是 CryZAb4，并分别于 12、16、16、16、20hrs进人稳定生长期。而 Bt菌
株HD．刀是在生长4小时后进入对数生长期，！2小时后进入稳定生长期。
2·＊【Cr）、蛋白分离纯化的研究
研究了 Bt菌株和在Ecoli中 Bt Cry蛋白的提取时机，先后分离纯化了印·ICa7。
Crymm、＊nlla巳＊nnel、吐2汕4蛋白和标准菌株＊ilAc（＊D一刀）蛋白，研究
表明在 ECCll中表达的蛋白均在培养 lershrs含量达到最高，CylAC晶体蛋白则是在培养
28～32hrs达到最高c将这几种蛋白经 FPLC纯化并进行了活性分析表明 CylAc在 sedml
处的洗脱峰活性较高，而在CO，基因ECOli中表达产物则是在IDellnd处有较高的活性。
3、Bt Cr）蛋白对棉铃虫幼虫毒性的研究
将上述 6种 Cy蛋白对棉铃虫进行初筛和两两正交组合的复筛．结果表明 CylAc和
CnZAb4对棉铃虫的毒性较强，LC。。分别为刀．sug／thl和415ghl；Cy1Ac＋Cn·ZAb4组
合的LC。；；为48．7twml，两者的共毒系数（C。TC）为121，具有一定的增效作用。其余
毒素对棉铃虫毒力的强弱依次是 Cryllel、Cryllas、CylCa7、CrylCbZ，这几种蛋白的
组合对棉铃虫没有增效作用。
4·Bt Cr3蛋白对玉米螟幼虫毒性的研究
将上述 6种 Cy蛋白对玉米螟进行初筛和两两正交组合的复筛，结果表明q、IAC。
Cn，Ilas。Cn，Ilel对玉米螟有很强的活性，将这三种蛋白进行正交组合对玉米螟进行生测，
发现k，IAc与C口llel、Cfl，ilas分别有明显增效作用、相加作用，用校正死亡率进行
SAS分析 C。IAc、C。·ilas、Cn，Ilel、CrvlAc＋Cryllel、CrylAc＋Cryllas的 LC。。分别
为：0，134、0500、0二23、0046、of98wi。CylAc＋Cry·Ilel和 CrvlAC＋Cryllas蛋白组
合有显著的增效作用，CTC．分别为 409和 106，在 LC。、LC。。水平 CylAC的毒力最
高。这些研究结果说明了CrylAc、Cryrllel、Cry，IAdellel以及CrvlAc＋Cryllas蛋白
组合对玉米螟有很强的杀虫活性，它们完?
Lepidoptera pests, such as cotton boll worm, beet army worm, and com borer worm, were very harmful to many crops and vegetables. Recently, resistance of cotton boll worm and beet army worm for chemicals was increasing quickly in the field, and it was very hard to control some pest by traditional methods. Leguminivora glycinivorella was the most important pest for soybean in north of China. But no formal report involved in the biocontrol using Bt
Bt, the best microbial insecticide, has been reported for control of cotton boll worm, beet worm, but the narrow choice of cry genes, for instance only crylAb or cry1 Ac, may induce the pest resistance.
In this study , different Cry protein expressed in E.coli. and the combinations between Cry proteins were screened against mentioned pests. It was very significant for construction of novel genetically engineered bacteria and transgenic plant for insect pest biocontrol.
1. Biological characteristics of Bt Cry protein
The solubility, digestion of Cry protein with trypsin and growth speed of E.coli. strain were researched respectively.
The solubility of CrylCa7, CrylCb2, Crylla8, CrylIe1, Cry2Ab4 proteins, expressed in E.coli. strains, and CrylAc from HD-73 strain were demonstrated that they were more soluble in Na2CO3 than in Ethanoloanmine by SDS-PAGE analysis.
Trypsin, the hydrdytic enzyme of insects, was crucial for digestion and toxicity of Cry protein. After reaction 6hrs, all the Cry protein were digested, and 55kDa proteins were obtain from CrylCa7 and Crylla8, 57kDa fragment were from CrylCb2, Cryllel and Cry2Ab4, but the 60kDa, peptide was maintained from CrylAc. If the concentration and reaction time were much more , all the Cry protein were degraded completely.
It was the first report of the digestion of Cryllel and CrylIa8 in the LB broth, the growth speed of all the E.coli. strains harboring the recombinant plasmids inserted cry gene, were slower than their host BL21. The over expression of cry gene maybe affect on the growth and development of host cell.
2. Purification of Bt Cry proteins
Expression time of cry protein in Ecoli strain was investigated. After induction by IPTG, 4~6hrs, the accumulation of Cry protein was the most, but prodution of CrylAc from HD-73 strain was the most after 28~32hrs. The result of FPLC showed that all of protoxins and toxins activated by tryspin could be well purified using elutin buffer at pH9.5
3. Bioassay of Cry protein against cotton boll worm larvae
The bioassay result demonstrated that Cry 1 Ac and Cry2Ab4were higher toxicity than other four Cry proteins, and LC50 was 31.5ug/ml and 415ug/ml respectively. Furthermore, the co-toxicity coefficient of Cry lac and Cry2Ab4 combination was 121% according to the LC50 48.7ug/ml of protein combination. It was means that Cry 1 Ac and Cry2Ab4, but other four Cry toxin, could synergize each other.
4. Bioassay of Cry protein against corn borer worm larvae
The result of bioassay indicated that Cry1Ac, Crylla8 and CrylIe1 were high toxicity, and the combination between the three protein, illustrated that CrylIe1+CrylAc combination were synergistic against corn borer worm larvae. LC50 of Cry1Ac, CrylIa8, CrylIe1, CrylAc+CrylIe1 and CrylAc+CrylIa8 was 0.134, 0.500, 0.223, 0.046, and 0.198ug/g respectively. Cotoxicity coefficients of CrylIe1+CrylAc and Crylla8+CrylAc were 409 and 106 separately. Cry1Ac was the highest among the Cry protein on the level of LC90 and LC95. Those bioassay results showed that CrylAc+CrylIe1 combination could be used for construction of engineered bacteria and transgenic plant, and meanwhile Cry1Ac as an enhancer protein could be combinated with other Cry protein for Insecticidal research.
5. Bioassay of Cry protein against beet army worm larvae
The larvae could be inhibited by all the Cry protein, and Cry1Ac and CrylCa7 combination was more toxic.
6. Bioassay of Cry protein against Leguminivora glycinivorella larvae
The result of bioassay of Cry protein to Leguminivora glycinivorella larvae, by two time select
引文
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