苏云金芽孢杆菌Cry1Ac杀虫晶体蛋白及其分子设计

cry1Ac编码的杀虫晶体蛋白是苏云金芽孢杆菌(Bt)产生的多种杀虫晶体蛋白中对鳞翅目昆虫有很高毒性的蛋白.第一个Cry1Ac杀虫晶体蛋白最早在库斯塔克亚种HD73中以伴胞晶体形式分离获得,其编码区为3 534 bp,编码蛋白分子量为133 kD,含1 178个氨基酸,等电点为4.84.自此以来,Cry1Ac杀虫晶体蛋白结构、功能以及应用研究一直是Bt杀虫晶体蛋白研究的重要方向.本文介绍了苏云金芽孢杆菌中应用最广泛的Cry1Ac杀虫晶体蛋白家族的结构、功能及其基因分类,并进一步就基于苏云金芽孢杆菌Cry1Ac杀虫晶体蛋白的基因工程研究做了分析,提出了持续利用BtCry1Ac杀虫晶体蛋白的一些见解.     

苏云金芽孢杆菌的cry2A芽孢期启动子和分子伴侣ORF1-ORF2对Cry11Aa蛋白表达的影响

[目的]分析苏云金芽孢杆菌的cry2A型芽孢期启动子对晶体蛋白Cry11Aa的协调作用和分子伴侣ORF1-ORF2对Cry11Aa表达的促进功能.[方法]3个包括cry11Aa编码区的重组质粒pHcy1、pHcy2和pHcy4被构建并电激转化到苏云金芽孢杆菌晶体缺陷株4Q7中,其中pHcy1质粒携带cry11Aa基因自身启动子和分子伴侣p19基因,pHcy2携带cry2A型芽孢期启动子和分子伴侣orf1-orf2基因,pHcy4质粒在pHcy1的上游插入了cry2A型芽孢期启动子和分子伴侣orf1-orf2基因.SDS-PAGE分析了Cry11Aa蛋白在各重组苏云金菌株中的表达情况,并通过生物测定确定了其对蚊虫的生物活性.[结果]SDS-PAGE结果表明,Cry11Aa蛋白在4Q7(pHcy1)和4QT(pHcy4)均获得了表达,在4Q7(pHcy2)中未检测到Cry11Aa蛋白,推测晶体蛋白Cry11A不能利用cry2A型启动子进行表达调控;Cry11Aa蛋白在等体积4Q7(pHcy4)培养液中的表达量是4Q7(pHcy1)菌株的1.25倍,暗示着分子伴侣ORF1-ORF2在某种程度上能提高Cry11Aa的蛋白表达量.4Q7(pHcy1)和4Q7(pHcy4)形成的Cry11Aa蛋白晶体的形状和大小相似,两者对致倦库蚊的生物活性没有明显差异,LC50s分别为59.33 ng/mL和66.21 ng/mL,.[结论]推测晶体蛋白Cry11A能否成功表达与其使用启动子的类型和两者的协调配合有关.分子伴侣ORF1-ORF2虽然在某种程度上能提高Cry11Aa的蛋白表达量,但对提高Cry11Aa蛋白的杀蚊毒力没有显著性帮助.     

苏云金杆菌辅助蛋白P19对杀虫晶体蛋白Cry11Aa表达的影响

苏云金杆菌以色列亚种的p19基因、cry11Aa基因和p20基因位于同一操纵子上,据推测辅助蛋白P19可能与Cry11Aa蛋白的晶体化相关。本研究利用穿梭载体pHT3101构建了两个重组质粒pHcy1和pHcy3,两质粒均携带cry11Aa基因,但后者完全缺失了cry11Aa基因上游的p19基因。将重组质粒电激转化至苏云金杆菌无晶体突变株4Q7中进行蛋白表达,SDS-PAGE结果表明在4Q7(pHcy1)和4Q7(pHcy3)中均能检测到正常表达的Cry11Aa蛋白,但单位体积培养液的Cry11Aa蛋白在辅助蛋白P19存在时的表达量明显高于其单独表达的表达量;透射电镜观察显示两菌株中的Cry11Aa蛋白形成了大小相近、形状相似的双梯形晶体;另外,生物测定结果表明重组菌株4Q7(pHcy1)和4Q7(pHcy3)对三龄致倦库蚊的杀虫活性没有显著性差异。该现象说明辅助蛋白P19的缺失对Cry11Aa蛋白的晶体形成和杀蚊活性没有影响,但P19作为分子伴侣在一定程度上帮助提高了Cry11Aa蛋白的表达水平。     

携带苏云金芽胞杆菌cry1Ac10基因重组杆状病毒的构建及其杀虫活性

用Bac-to-Bac系统,构建了包含极晚期基因ph启动子驱动的带有全长苏云金芽胞杆菌cry1Ac10基因和完整多角体基因的重组质粒pFCP,用该重组质粒感染昆虫Sf9细胞,得到了带有多角体和能够表达cry1Ac10基因的重组杆状病毒vFcph,并在昆虫细胞中表达了Cry1Ac10蛋白。同时构建了含cry1Ac10的穿梭载体．pHTC,并分别转化大肠杆菌、枯草杆菌和苏云金杆菌晶体缺陷型菌株,结果表明此三种工程菌均表达了分子量为133．3kDa的原毒素蛋白,其中在苏云金芽胞杆菌中的表达量最高。生物测定表明重组杆状病毒vFcph的表达产物具有杀虫活性,能增加杆状病毒力,加快杆状病毒杀虫速度,说明利用杆状病毒极晚期基因启动子驱动苏云金芽胞杆菌杀虫晶体蛋白表达,从而改善杆状病毒的杀虫特性是可行的。     

携带苏云金芽胞杆菌cry1Ac10基因重组杆状病毒的构建及其杀虫活性

用Bac-to-Bac系统,构建了包含极晚期基因ph启动子驱动的带有全长苏云金芽胞杆菌cryIAc10基因和完整多角体基因的重组质粒pFCP, 用该重组质粒感染昆虫Sf9细胞,得到了带有多角体和能够表达cry1Ac10基因的重组杆状病毒vFcph,并在昆虫细胞中表达了Cry1Ac10蛋白.同时构建了含cry1Ac10的穿梭载体pHTC,并分别转化大肠杆菌、枯草杆菌和苏云金杆菌晶体缺陷型菌株,结果表明此三种工程菌均表达了分子量为133.3kDa的原毒素蛋白,其中在苏云金芽胞杆菌中的表达量最高.生物测定表明重组杆状病毒vFcph的表达产物具有杀虫活性,能增加杆状病毒力,加快杆状病毒杀虫速度,说明利用杆状病毒极晚期基因启动子驱动苏云金芽胞杆菌杀虫晶体蛋白表达,从而改善杆状病毒的杀虫特性是可行的.     

苏云金芽胞杆菌Rpp39杀虫晶体蛋白基因的鉴定及cry2Aa12基因的克隆表达

[目的]本研究的目的是分析从四川生态条件下分离的苏云金芽胞杆菌Rpp39菌株的特性,从分子水平上揭示该菌株对鳞翅目高毒力的原因;进一步从中分离克隆cry2Aa基因,并对其进行初步的表达研究.[方法]本研究主要采用扫描电镜观察、PCR-RFLP鉴定法和SDS-PAGE分析法研究菌株的特性;采用PCR直接克隆法克隆cry2Aa全长基因,并亚克隆到原核表达载体pET-30a中,构建重组表达质粒pET-2Aa,再转入受体菌E.coli.BL21(DE3)中进行诱导表达;采用室内生物测定法测定表达产物对小菜蛾和水稻二化螟的毒力.[结果]经扫描电镜观察菌株Rpp39主要产生菱形、方形和圆形3种伴胞晶体;SDS-PAGE分析表明主要产生130 kDa和60 kDa左右2种蛋白;经PCR-RFLP鉴定,该菌株含有cry1Aa、cry1Ab、cry1Ac、cry1Ia和cry2Aa五类杀虫晶体蛋白基因;1种cry2Aa类杀虫晶体蛋白全长基因被克隆,序列分析显示该基因的开放阅读框(ORF)为1902 bps,编码由634个氨基酸组成的蛋白质,氨基酸序列与Cry2Aa1蛋白同源性为99.7%,被国际Bt杀虫晶体蛋白基因命名委员会命名为cry2Aa12.重组表达质粒pET-2Aa在E.coli BL21(DE3)中,经IPTG诱导能正常表达,SDS-PAGE电泳验证含有65 kDa表达蛋白.生物活性测定表明表达的包涵体蛋白对小菜蛾和二化螟具有杀虫活性,LC50分别为5.4 μg/mL和22.3μg/mL.[结论]菌株Rpp39及从中分离克隆的cry2Aa12基因来自四川生态条件,丰富了菌株及基因的资源,在资源积累方面具有重要意义.     

棉铃虫中肠钙粘蛋白、氨肽酶N及碱性磷酸酯酶的抗血清对Cry1Ac和Cry2Aa毒力的影响

【目的】Cry1A和Cry2A类Bt蛋白通过特异性地与昆虫中肠上的受体蛋白结合而发挥杀虫作用,现已广泛应用于转基因抗虫作物。本研究旨在进一步明确Cry2A类蛋白的作用机制和Cry1A受体蛋白在Cry2A发挥毒力中的作用。【方法】本研究首先提取了棉铃虫Helicoverpa armigera的BBMV,制备了钙粘蛋白(CAD)、氨肽酶N(APN)和碱性磷酸酯酶(ALP)3种受体蛋白的抗体和抗血清;然后,利用Western blot检测BBMV上这3种受体蛋白后,利用抗体封闭技术比较了敏感棉铃虫和Cry1Ac抗性棉铃虫(BtR)中3种受体蛋白的抗血清对Cry1Ac和Cry2Aa毒力的影响。【结果】对敏感品系棉铃虫,这3种已知的Cry1Ac受体蛋白抗血清显著地降低了Cry1Ac和Cry2Aa的毒力。其中APN抗血清对Cry1Ac毒力的影响最大,棉铃虫幼虫的死亡率降低了84.44%;ALP抗血清对Cry2Aa的毒力影响最大,棉铃虫幼虫死亡率比对照降低了71.04%。Cry1Ac对Cry1Ac抗性棉铃虫(BtR)的毒力显著降低,Cry2Aa的毒性也减弱。在Cry1Ac抗性棉铃虫(BtR)中,3种受体抗血清对Cry1Ac的影响比在敏感棉铃虫中的影响小,尤其是CAD和APN抗血清对Cry1Ac毒力的抑制率显著低于在敏感棉铃虫中的抑制作用;CAD和ALP抗血清对Cry2Aa毒力的影响与在敏感棉铃虫中的影响差异不显著,但APN抗血清可以显著降低Cry2Aa对Cry1Ac抗性棉铃虫(BtR)的毒力。【结论】棉铃虫CAD,APN和ALP不仅参与了Cry1Ac的杀虫过程,也对Cry2Aa毒力有一定的影响,而且这3种蛋白可能与棉铃虫对Cry1Ac和Cry2Aa产生抗性及交互抗性相关。     

Cry1Ac基因载体构建及其在枯草芽孢杆菌中的杀虫活性表达

根据苏云金芽孢杆菌Bacillus thuringiensis HD-73基因Cry1Ac和枯草芽孢杆菌Bacillus subtilis木糖诱导型启动子PxylR序列, 分别设计2对特异引物Cry1Ac F/R和Pxy F/R,扩增获得了完整的启动子PxylR和Cry1Ac基因序列,进一步以上述产物混合物为模板,以Pxy F/Cry1Ac R作引物进行重迭PCR,获得了载体PxylR-Cry1Ac,经SphⅠ和BamHⅠ完全酶切后,将PxylR-Cry1Ac插入大肠杆菌-苏云金芽孢杆菌穿梭载体pHT315,重组表达质粒pCry1Ac315转化枯草芽孢杆菌感受态细胞。工程菌株质粒酶切电泳分析、SDS-PAGE电泳分析和杀虫生物活性测定结果证实了Cry1Ac基因的导入及其在枯草芽孢杆菌JAAS01D中的有效表达。     

苏云金杆菌辅助蛋白P20对杀虫晶体蛋白Cry1Ab表达的影响

苏云金杆菌(Bacillus thuringiensis, 简称Bt)杀虫晶体蛋白Cry1Ab因其C半端缺少了一段含4个半胱氨酸的氨基酸序列而导致蛋白的不稳定,报道苏云金杆菌辅助蛋白P20帮助Cry1Ab蛋白的表达及晶体的形成。利用穿梭载体pHT3101构建3个表达质粒,即pT1B、pP1B和pDP1B,3个质粒都含有cry1Ab基因,不同在于pT1B没有p20基因,pP1B含有p20全基因,而pDP1B不仅含有p20全基因,且在p20基因前插入cry1A?启动子。分别将这3个表达质粒经电转化到苏云金杆菌晶体缺陷型菌株CryB中,获得转化菌株T1B、P1B和DP1B。Western blot表明cry1Ab基因在这3株菌中均表达了130 kD的蛋白,部分降解为大约60 kD的蛋白。蛋白定量分析显示,3株菌130 kD蛋白量的比为1∶1.4∶1.5,降解后的60 kD蛋白量的 比为1∶1.1∶1.6,Cry1Ab蛋白总量的比为1∶1∶2∶1.6。镜检发现,Cry1Ab在3株菌中都形成典型的菱形晶体,其晶体大小为T1B Helicoverpa armigera）均具有明显的杀虫活性,三者的LC₅₀差异不显著。研究表明,P20对cry1Ab基因的表达和晶体形成均有帮助,P20表达量的多少可能是导致Cry1Ab蛋白最终产量有所不同的因素。     

苏云金芽胞杆菌中荧光分析融合杀虫晶体蛋白的形成

为研究苏云金芽胞杆菌杀虫晶体蛋白在细胞中的定位及在细胞中的形成, 构建了Cry1Ac-GFP融合蛋白, 大小约为160 kD. 将携带cry1Ac启动子的cry1Ac-gfp融合基因片段克隆到pHT304载体上, 获得融合表达载体pHTcry1Ac-gfp. pHTcry1Ac-gfp转化到无晶体突变株HD-73 cry-中, 获得融合表达菌株HD-73-(pHTcry1Ac-gfp). gfp基因通过同源重组插入到HD-73内源大质粒pHT73上cry1Ac基因的3′端, 获得原位融合表达菌株HD-73Φ(cry1Ac-gfp)3534. 激光共聚焦显微镜和Western杂交分析表明, 不对称隔膜形成时, HD-73-(pHTcry1Ac-gfp)和HD-73Φ(cry1Ac-gfp)3534细胞中检测到Cry1Ac-GFP融合蛋白的表达. 融合蛋白颗粒在细胞中的聚集存在一定的极性, 分布于母细胞不对称隔膜附近. Cry1Ac-GFP和Cry1Ac蛋白对小菜蛾的杀虫活性在95%置信区间内没有明显差异.     

昆虫类钙粘蛋白与Bt Cry1A蛋白之间的相互作用

类钙粘蛋白(cadherin-likeprotein)位于昆虫中肠刷状缘膜囊泡(brushbordermembranevesicles,BBMV)上,是苏云金芽孢杆菌(Bacillusthuringiensis,Bt)产生的杀虫晶体蛋白(BtCry蛋白)的主要受体之一。它能够与BtCry蛋白结合,引起细胞膜的渗透性发生改变,促进BtCry蛋白对敏感昆虫的毒杀作用。类钙粘蛋白基因的突变还能导致敏感昆虫对BtCry蛋白产生抗性。因此,研究昆虫类钙粘蛋白与BtCry蛋白之间的相互作用,将有助于揭示BtCry蛋白杀虫作用机理。文章对昆虫类钙粘蛋白种类、结构特征、在昆虫体内的分布、及其与BtCry蛋白之间的相互作用等方面的研究现状进行详细论述。     

Carboxy-terminal extension effects on crystal formation and insecticidal properties of Cry15Aa

Cry15Aa protein, produced by Bacillus thuringiensis serovar thompsoni HD542, in a crystal together with a 40 kDa accompanying protein, is one of a small group of non-typical, less well-studied members of the Cry family of insecticidal proteins, and may provide an alternative for the more commonly used Cry proteins in insect pest management. In this study we examined the role of the C-terminal part of Cry15Aa and of the 40 kDa protein in crystal formation in recombinant B. thuringiensis. The contribution of the 40 kDa protein and of the Cry15Aa carboxy-terminal sequence for crystal formation, crystal solubilization, and insecticidal properties was assessed. No significant differences in toxicity against Cydia pomonella, before or after in vitro solubilization of crystal-spore preparations, were found. Although the 40 kDa protein significantly contributes to in vitro solubility and in vivo crystal formation of Cry15Aa, no direct evidence for involvement of the 40 kDa protein in toxicity of Cry15Aa was found.     

The synergistic activity between Cry1Aa and Cry1c from Bacillus thuringiensis against Spodoptera exigua and Helicoverpa armigera

AIMS: To investigate the interaction between two crystal proteins, Cry1Aa and Cry1C, for future development of biopesticides based on Bacillus thuringiensis, toxicities of the two individual proteins and in combinations have been determined against Spodoptera exigua and Helicoverpa armigera larvae, and synergism between the proteins has been evaluated using synergistic factor. METHODS AND RESULTS: SDS-PAGE showed that Cry1Aa and Cry1C proteins could be expressed in acrystalliferous B. thuringiensis 4Q7 strain, with molecular weights of 135 and 130 kDa respectively. The bioassay results indicated a synergistic activity between Cry1Aa and Cry1C against S. exigua and H. armigera, and the highest toxicities could be observed in the combination of Cry1Aa and Cry1C at a ratio of 1 : 1. CONCLUSION: The two toxins, Cry1Aa and Cry1C, interact synergistically to exhibit higher toxicity against S. exigua and H. armigera. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the investigation on the synergistic activity between two B. thuringiensis Cry1 toxins. It can be applied to the rational design of new generations of B. thuringiensis biopesticides and to strategies for management of resistant insects.     

一些化学物质对苏云金芽孢杆菌Cry1Ac毒素与昆虫离体细胞相互作用的影响

为探讨苏云金芽孢杆菌Bacillus thuringiensis（Bt）杀虫晶体蛋白与昆虫细胞的相互作用,以Bt Cry1Ac毒素和对该毒素敏感的粉纹夜蛾Trichoplusia ni离体细胞BTI-TN-5B1-4为材料,研究了一些化学物质对Cry1Ac毒素与昆虫离体细胞相互作用的影响.结果表明：N-糖基化抑制剂衣霉素、蛋白质合成抑制剂放线菌酮、胞吞作用抑制剂莫能菌素和胰蛋白酶预处理,都能不同程度地提高BTI-TN-5B1-4细胞对Cry1Ac毒素的敏感性,其中胰蛋白酶预处理的作用最明显;而N-乙酰半乳糖胺不能抑制Cry1Ac毒素对这种离体细胞的毒力.     

Expression and crystallization of a Cry3Aa–Cry1Ac chimerical proteinof Bacillus thuringiensis

The insecticidal Cry1 proteins of Bacillus thuringiensis form a typical bipyramidal parasporal crystal and their protoxins contain a highly conserved C-terminal region. A chimerical gene was constructed with the coding regions of the Cry3Aa protein's toxic domain, and of the Cry1Ac protoxin's C-terminal fragment. This chimerical construction expressed a truncated (70kDa) protein in the acrystalliferous strain 4Q7 of B. thuringiensis, assembled in spherical to amorphous parasporal crystals. This protein was recognized only by antibodies raised against the Cry3Aa protein. When the protease-deficient mutant BL21 of Escherichia coli was transformed with the same chimerical construction, a complete (140kDa) chimerical protein was expressed. However, the formation of a crystalline inclusion was unclear. This protein was recognized by antibodies raised against the proteins Cry1Ac and Cry3Aa. Both chimerical proteins showed toxicity against larvae of Leptinotarsa texana, being much more active when expressed truncated in B. thuringiensis. These results suggest that the formation of bipyramidal crystals requires more than just the presence of the C-terminal region of Cryl protoxins. They also suggest that proteolysis plays an important role during the post-translational processing of Cry proteins.     

Interaction between Cry9Ca and two Cry1A delta-endotoxins from Bacillus thuringiensis in larval toxicity and binding to brush border membrane vesicles of the spruce budworm,Choristoneura fumiferana Clemens

A genetically altered variant of Cry9Ca from Bacillus thuringiensis shows high potency against the spruce budworm, Choristoneura fumiferana Clemens. Its activity, as measured by feeding inhibition in frass-failure assays, is estimated to be four to seven times greater than B. thuringiensis subsp. kurstaki HD-1, the strain currently used in commercial products to control this insect. Bioassays against budworm of mixtures of the modified Cry9Ca and two of the Cry1A endotoxin proteins produced by HD-1 show neither synergism nor antagonism. Experiments with brush border membrane vesicles from budworm midgut revealed that Cry9Ca and the Cry1A toxins share a common binding site and that bound Cry9Ca can be displaced from the membrane to some extent by the Cry1A toxins. However, it is uncertain whether the binding site is actually the receptor molecule or a membrane protein associated with pore formation.     

Single cysteine substitution in Bacillus thuringiensis Cry7Ba1 improves the crystal solubility and produces toxicity to Plutella xylostella larvae

Many Bacillus thuringiensis isolates have no demonstrated toxicity against insects. In this study, a novel holotype crystal protein gene cry7Ba1 was isolated from a 'non-insecticidal'B. thuringiensis strain YBT-978. The Cry7Ba1 protein showed high toxicity against Plutella xylostella larvae after the crystals were dissolved at pH 12.5, suggesting that the 'non-insecticidal' properties of this protein were due to insolubility in the normal insect midgut pH environment. After the C-terminal half of Cry7Ba1 was replaced by that of Cry1Ac or Cry1C proteins, the recombinant protein inclusions could be dissolved at pH 9.5, and exhibited high toxicity against P. xylostella larvae. This result proved the insolubility of Cry7Ba1 crystal was determined by the structure of its C-terminal half. Further, six mutations were constructed by substituting cysteine residues with serine. Solubility studies showed that the crystals from mutants C697S, C834S and C854S could be dissolved at lower pH (10.5, 9.5 and 11.5 respectively). Bioassays showed that crystals from mutant C834S were toxic to P. xylostella larvae. Our discoveries suggest that a single cysteine residue located in the C-terminal half of the protein determines the solubility and toxicity of some nontoxic crystal proteins. This study provides a strategy to isolate novel insecticidal crystal protein genes from 'non-insecticidal'B. thuringiensis strains.     

Recombinant Cry3Aa has insecticidal activity against the Andean potato weevil, Premnotrypes vorax

The Andean potato weevil, Premnotrypes vorax, an insect of the order Coleoptera, is a major cause of damage to potato crops in the Andean regions of South America. The insecticidal Cry proteins from Bacillus thuringiensis are useful biological pesticides, and some are toxic to Coleopteran insects. We overexpressed recombinant, histidine-tagged Cry3Aa protein in Escherichia coli host cells. The recombinant protein was solubilized at high pH with urea, purified using Ni(2+)-nitrilo-triacetic acid affinity resin, and dialysed to lower pH and remove urea. Bioassays were performed with an insect media whose surface was spread with 70 microgram/mL purified native or recombinant toxins. First instar larvae exposed to toxin treated media for 5 days exhibited mortalities from 57% (native Cry3Aa) to 52% (recombinant Cry3Aa). Purified native and recombinant Cry3Aa proteins appeared to be equally toxic to the Andean potato weevil.     

Large scale production of Bacillus thuringiensis PS149B1 insecticidal proteins Cry34Ab1 and Cry35Ab1 from Pseudomonas fluorescens

The 14kDa (Cry34Ab1) and 44kDa (Cry35Ab1) binary insecticidal proteins are produced naturally by Bacillus thuringiensis PS149B1 as parasporal inclusion bodies. Here, we show production of these two insecticidal proteins in recombinant Pseudomonas fluorescens and their subsequent purification to near homogeneity to provide large quantities of protein for safety-assessment studies associated with the registration of transgenic corn plants. The gene sequence specific for each protein was expressed in P. fluorescens and fermented at the 75-L scale. For Cry34Ab1, the protein accumulated as insoluble inclusion bodies, and was purified by extraction directly from the cell pastes at pH 3.4 with a sodium acetate buffer, selective precipitation at pH 7.0, and differential centrifugation. For Cry35Ab1, the protein was extracted from the purified inclusion bodies with sodium acetate buffer (pH 3.5) containing 0.5M urea, followed by diafiltration. No chromatography steps were required to produce over 30g of lyophilized protein powder with purity greater than 98%, while retaining full insecticidal activity against Western corn rootworm larvae. The proteins were further characterized to assure identity and suitability for use in safety-assessment studies.