31株苏云金芽孢杆菌杀虫晶体蛋白基因型鉴定及表达产物研究

利用已建立的苏云金芽孢杆菌cry基因的PCRRFLP鉴定体系,鉴定了31株Bt菌株的cry基因类型,并进行了SDSPAGE分析和杀虫生物活性测定。研究表明：25株含cry1基因,表达蛋白130～150kD;其中16株含有对鞘翅目和鳞翅目害虫皆有活性的cry1I基因,其表达蛋白为81kD;15株同时含有cry1和cry2基因(13株表达蛋白约为60kD);10株含有未知待定基因;6株不含所鉴定的cry基因(其中2株有表达产物)。室内生物测定表明：cry1、cry2基因表达的菌株对鳞翅目害虫具有高杀虫活性,7株对舞毒蛾和膜翅目——杨叶蜂幼虫具有较高杀虫活性;含有cry1Aa\,cry1Ac\,cry2或cry1Ab\,cry1Ac\,cry2基因组合的菌株对棉铃虫幼虫均显示杀虫活性,其中6、12、30号菌株毒力最强。不含上述cry基因的菌株均无杀虫活性。以上结果证明,通过cry基因类型鉴定和表达产物的SDSPAGE分析可以预测菌株的杀虫活性。     

利用绿色荧光蛋白基因gfp研究芽胞杆菌的启动子活性

利用绿色荧光蛋白基因gfpmut3,分别标记苏云金芽胞杆菌(Bacillus thuringiensis)的cry3A启动子Pcry3A、BtI_BtII启动子PBtI_BtII和来自蜡状芽胞杆菌特异启动子P44-12以研究其表达差异。其中,Pcry3A和PBtI_BtII分别与gfpmut3构成融合基因,以调控gfpmut3在苏云金芽胞杆菌中的表达。将重组质粒pGFP_304(含P44-12)、pGFPExpA(含Pcry3A_ gfpmut3融合基因)和pGFPExpB(含PBtI_BtII_ gfpmut3融合基因)分别导入大肠杆菌(Escherichia coli)和苏云金芽胞杆菌后发现,P44-12和PBtI_BtII在大肠杆菌与苏云金芽胞杆菌中均可表达gfpmut3,其中PBtI_BtII在大肠杆菌中具有极强的启动基因表达的能力。而Pcry3A不能启动gfpmut3在大肠杆菌中表达,在苏云金芽胞杆菌中启动的gfpmut3表达的荧光强度也较弱。进一步通过荧光显微镜和生物活性检测器对含重组质粒pGFP_304、pGFPExpA和pGFPExpB的转化子分别进行荧光检测及微量热检测。结果表明,3种启动子驱动下的gfpmut3基因均可在苏云金芽胞杆菌无晶体突变株BMB171中表达并检测得到不同的发光类型。微量热法检测发现P44_12和PBtI_BtII启动gfpmut3表达的代谢热低于Pcry3A驱动gfpmut3表达的代谢热。     

苏云金芽胞杆菌YBT1520杀虫晶体蛋白基因的属性

通过Southern杂交发现高毒力苏云金芽胞杆菌(\%Bacillus thuringiensis)\% YBT1520菌株含有两个杀虫晶体蛋白基因片段,其5’末端所在HindⅢ片段分别为68kb和46kb,它们对应的基因分别命名为cry218和cry46。经PCR鉴定,该菌含有cry1Aa\,cry1Ab和cry1Ac基因,以及cry2基因,其中cry218属于cry1Ac。分析了cry218基因4190bp的核苷酸序列,在杀虫晶体蛋白基因分类系统中被命名为cry1Ac10。结合Southern杂交和PCR结果可判断3个cry1A基因的拷贝数不同,其中cry1Ac拷贝数最高,YBT1520菌株与其它库斯塔克亚种的杀虫晶体蛋白基因所在限制性内切酶位置明显不同。     

苏云金芽胞杆菌杀虫晶体蛋白基因cry1Ab16的克隆及表达

通过对已知cry1类基因以及已发表的cry1Ab的序列进行分析,分别设计了引物P1、P2、P3和P4,首次从无晶体的芽胞杆菌AC11中扩增到一个苏云金芽胞杆菌杀虫晶体蛋白（Insecticidal crystal protein, ICP）cry1Ab类基因。测序结果显示该基因与已知的cry1Ab1基因有8个核苷酸不同,编码的蛋白有7个氨基酸差异。此基因已登录GenBank,并命名为新亚型基因cry1Ab16 (Ac. NO. AF375608)。Southern杂交结果进一步证实该基因存在于菌体的质粒上。将cry1Ab16基因克隆到Escherichia coli表达载体pQE30上并转化E. coli M15。Western印迹分析表明,E. coli M15表达了130 kD的Cry1Ab16蛋白,但此蛋白不稳定,大部分降解成65 kD的蛋白。将表达Cry1Ab16 蛋白的大肠杆菌用涂布法对三龄小菜蛾（Plutella xylostella）毒力测定,其LC50为258.3mg/L;对其他夜蛾科害虫的生长发育也有明显的抑制作用。     

苏云金芽孢杆菌分子伴侣串联基因p19-p29的克隆和表达载体的构建

根据已知序列设计一对PCR引物(ORF5S,ORF3N),可从cry2Aa或cry2Ac操纵子中扩增出包含串联分子伴侣基因p19p29的DNA片段,预期大小分别为16kb和20kb。对150株苏云金芽孢杆菌菌株进行PCR检测,从26株中获得了大小为16kb的扩增片段,但未获得20kb的片段。这表明cry2Aa型操纵子p19p29基因存在较广泛,而cry2Ac型较罕见。将来自Y2菌株的16kb片段回收,通过一系列亚克隆,最终构建成一个含有p19p29串联基因的Bt表达载体,为进一步研究p19p29串联基因的功能奠定了基础。     

双价杀虫蛋白基因在荧光假单胞菌中的表达及增效

利用广宿主质粒载体pJMS6αlac将苏云金芽胞杆菌(Bacillus thuringiensis)杀虫晶体蛋白基因cry1Ac和cry2Aa基因分别及一起进行克隆,将重组质粒导入能在多种作物上定殖、对植物病菌有良好抑菌和防治作用的荧光假单胞菌(Pseudomonas fluorescens)P303菌株,分别得到工程菌株IPP101、IPP201和IPP202。PCRRFLP和Southern blot检测均证明目的基因已经导入了工程菌。SDSPAGE电泳显示工程菌中存在明显的Cry1Ac蛋白带;透射电镜观察发现含cry1Ac基因的两个菌株IPP101和IPP202中杀虫蛋白形成了典型的菱形晶体和蛋白包含体,而在野生P303菌株中均无这些结构。这些结果说明,工程菌中cry1Ac基因得到了很好表达。室内杀虫试验表明：工程菌对棉铃虫初孵幼虫的致死中浓度(LC50),只含cry1Ac的IPP101为000812mL/g饲料,只含cry2Aa的IPP201为002604mL/g饲料,含双基因的IPP202为000186mL/g饲料;HD73为000170mL/g饲料。cry1Ac和cry2Aa双基因表达产物具有显著增效作用,共毒系数达3328。     

苏云金芽胞杆菌cry26Aa和cry28Aa基因共表达可在芽胞外壁内侧形成伴胞晶体

苏云金芽胞杆菌幕虫亚种的伴胞晶体在预芽胞外壁内侧形成,呈现晶体芽胞粘连的现象。根据已发表的cry26Aa1和cry28Aa1基因序列设计引物,从苏云金芽胞杆菌幕虫亚种T02中扩增得到cry26Aa和cry28Aa基因,通过穿梭载体将这两个基因分别和同时转化到苏云金芽胞杆菌无晶体突变株BMB171后,透射电镜下可在芽胞外壁内侧和外侧同时观察到伴胞晶体,而单独表达时可在芽胞外壁外侧观察到伴胞晶体。结果表明,伴胞晶体在芽胞外壁内侧表达不单独依赖于启动子的时空调控,可能还受到晶体蛋白相互作用的影响。     

苏云金芽孢杆菌杀虫晶体蛋白基cry3A在鳞翅目特异菌株中的表达及杀虫特性

将对鞘翅目昆虫有特异毒性的苏云金芽孢杆菌cry3A基因电转化到只对鳞翅目昆虫有毒性的苏云金芽孢杆菌野生型菌株YBT803-1中,获得转化了BMBY-001。SDS-PAGE分析及镜检结果表明,cry3A基因可在该菌株中高效表达,但出发菌株中原有的cry1Ab、cry1Ac及cry2的表达则受到不同程度的影响。生物测定结果显示,转化子BMBY-001对柳蓝叶甲(鞘翅目)具有较高毒力,LC50为0.413μL／mL(浸叶法),对小菜蛾(鳞翅目)的毒力比野生受体菌YBT803-1有所降低,LC50值为3.319μL／mL。     

利用苏云金芽胞杆菌转座子Tn4430构建含cry1Ac10基因的解离载体

将cry1Ac10基因和苏云金芽胞杆菌的复制起始区连 接在一起,并在其两侧按相同方向各连接一个来自苏云金芽胞杆菌转座子Tn4430的解离位点,构成转移单位。再将革兰氏阳性细菌的抗性标记基因和大肠杆菌克隆载体pUC19与之连接,获得含cry1Ac10基因的解离载体pBMB801。将其转化苏云金芽胞杆菌无晶体突变体,再导入含解离酶基因的辅助质粒pBMB1200。在解离酶作用下,两个解离位点间发生重组,消除基在操作中的抗性标记基因等非必需基因片段,获得仅保留有完整cry1Ac10基因和来 自苏云金芽胞杆菌质粒复制起始区,在无抗生素选择压力下能稳定遗传的重组质粒pBMB801B 。     

苏云金芽孢杆菌的特异性结合蛋白研究

用3 2 P分别标记 3 0 8bpcry1A上游和 65 0bpcry1C上游片段 ,并将标记后的DNA与不同苏云金芽孢杆菌菌株的细胞粗蛋白进行凝胶阻滞反应。结果表明 ,cry1A和cry1C上游均能被苏云金芽孢杆菌库斯塔克亚种 (Bacillusthuringinensis subsp .kurstaki)的细胞粗蛋白特异性结合 ,而同一cry1基因上游序列可被不同多肽特异或非特异性竞争结合 ,不同的cry1基因上游序列也能同时被一种蛋白结合。说明苏云金芽孢杆菌某些特异细胞蛋白参与了cry1基因上游序列的转录调控作用 ,而不同的调节因子可能会竞争同一结合位点。库斯塔克亚种和鲇泽亚种 (B .thuringinensis subsp .aizawai)所含特异细胞蛋白在种类和作用上都有差异。     

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.     

Characterization of <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis> Strain DOR4 Toxic to Castor Semilooper <Emphasis Type="Italic">Achaea janata</Emphasis>: Proteolytic Processing and Binding of Toxins to Receptors

We have isolated a strain of Bacillus thuringiensis (Bt) from Indian soil samples that was shown to be toxic to Achaea janata larvae. The isolate, named B. thuringiensis DOR4, serotypically identified with the standard subspecies kurstaki (H3a3b3c) and produced bipyramidal inclusions along with an amorphous type. Although the plasmid pattern of DOR4 was different from that of the reference strain, a crystal protein profile showed the presence of two major bands (130 and 65 kDa) similar to those of Bt subsp. kurstaki HD-1. To verify the cry gene content of DOR4, triplex PCR analysis was performed; it showed amplification of the cry1C gene in addition to cry1Aa, cry1Ac, cry2A, and cry2B genes, but not the cry1Ab gene. RT-PCR analysis showed the expression of cry1Aa and cry1Ac genes. In vitro proteolysis of DOR4 protoxin with midgut extract generated products of different sizes. Zymogram analysis of DOR4 protoxin as substrate pointed to a number of distinct proteases that were responsible for activation of protoxins. Furthermore, toxin overlay analysis revealed the presence of multiple toxin-binding proteins in midgut epithelium. Based on all these characterizations, we suggest that the Bt DOR4 strain can be exploited for an A. janata control program.     

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.     

Isolation and Characterization of a Bacillus thuringiensis ssp. kurstaki Strain Toxic to Spodoptera exigua and Culex pipiens

A strain of Bacillus thuringiensis with dual toxicity was isolated from Korean soil samples and named K2. K2 was determined as ssp. kurstaki (H3a3b3c) by serological test and produced bipyramidal-shaped parasporal inclusions. The plasmid and protein profiles of B. thuringiensis K2 were different from those of the reference strain, ssp. kurstaki HD-1. To verify gene type of B. thuringiensis K2, PCR analysis with specific cry gene primers was performed. The result showed that B. thuringiensis K2 had cry1Aa, cry1Ab, cry1C, and cry1D type genes, whereas ssp. kurstaki HD-1 had cry1Aa, cry1Ab, cry1Ac, and cry2 type genes. In addition, B. thuringiensis K2 had high toxicity against Spodoptera exigua and Culex pipiens, whereas B. thuringiensis ssp. kurstaki HD-1 does not have high toxicity against these two insect species. Received: 19 January 2001 / Accepted: 21 February 2001     

Vegetative insecticidal protein enhancing the toxicity of Bacillus thuringiensis subsp kurstaki against Spodoptera exigua

AIMS: The objective of this work was to enhance the insecticidal activity or widen the pesticidal spectrum of a commercial Bacillus thuringiensis strain YBT1520. METHODS AND RESULTS: A vegetative insecticidal protein gene vip3Aa7, under the control of its native promoter and cry3A promoter, was subcloned into B. thuringiensis acrystalliferous BMB171 to generate BMB8901 and BMBvip respectively. It was found that the amount of Vip3Aa7 protein produced by BMBvip was 3.2-fold more than that produced by BMB8901. Therefore, the vip3Aa7 gene under the control of cry3A promoter was transformed into strain YBT1520. The toxicity of the resulting strain BMB218V against Spodoptera exigua was 10-fold more than that of YBT1520, and that the toxicity of BMB218V against Helicoverpa armigera retained the same level as that of strain YBT1520. CONCLUSIONS: Strain YBT1520 obtained high toxicity against S. exigua after it was transformed and expressed the foreign vip3Aa7 gene. SIGNIFICANCE AND IMPACT OF THE STUDY: Commercial B. thuringiensis strain YBT1520 has high toxicity against H. armigera and Plutella xylostella, but almost no activity against S. exigua, which is a major crop pest in China. This work provides a new strategy for widening the activity spectrum of B. thuringiensis against agriculture pests.     

Engineered Bacillus thuringiensis GO33A with broad insecticidal activity against lepidopteran and coleopteran pests

A recombinant plasmid pSTK-3A containing cry3Aa7 gene encoding a coleopteran-specific insecticidal protein was constructed and introduced into wild Bacillus thuringiensis subsp. aizawai G03, which contained cry1Aa, cry1Ac, cry1Ca, and cry2Ab genes and was highly toxic to lepidopteran insect pests. The genetically engineered strain were named G033A. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot analysis demonstrated that the cry3Aa7 gene was expressed normally and produced a 67 kDa protein in G033A, and the flat rectangular crystals of Cry3Aa7 toxin protein was observed under scanning electron microscope. The recombinant plasmid was maintained in bacteria cultured for 180 generations in culture media containing no antibiotics. Synthesis of the Cry3Aa7 toxin conferred high and broad toxicity to the recombinant strain G033A against coleopteran order, elm leaf beetle (Pyrrhalta aenescens) (LC₅₀ 0.35 mg/ml), for which the parental strain G03 was not toxic. Both the parental strain G03 and recombinant strain G033A showed strong insecticidal activity to lepidopteran pests, beet armyworm (Spodoptera exigua), diamondback moth (Plutella xylostella), and cotton bollworm (Helicoverpa amigera), respectively. The lethal concentration 50% (LC₅₀) of G033A against S. exigua, P. xylostella, and H. amigera was 4.26, 0.86, and 1.76 μg/ml, respectively.     

苏云金芽孢杆菌4.0718菌株的杀虫晶体蛋白基因分析

根据苏云金杆菌(Bacillus thuringiensis)cry1、cry2和cry3型基因的保守区分别设计了3对通用引物Un1(d)/Un1?、Un2(d)/Un2?和Un3(d)/Un3?,以Bt4.0718菌株质粒DNA为模板进行PCR扩增,通过扩增产物片段的分子量大小来确定该菌株所含有的杀虫晶体蛋白基因类型。随后根据上述3类cry基因的高变区设计特异引物再次进行PCR鉴定。结果表明:Bt4.0718菌株含有cry1Aa、cry1Ab、cry1Ac、cry1Cb、cry2Ac和新基因cry4.5等6种基因类型。这一结果为利用该菌株构建高效广谱杀虫工程菌提供了客观依据。     

Isolation of a strain of Bacillus thuringiensis ssp. kurstaki HD-1 encoding δ-endotoxin Cry1E

A strain of Bacillus thuringiensis, STB-1, toxic against Spodoptera exigua , was isolated. Bacillus thuringiensis STB-1 produced bipyramidal inclusions and reacted with the H antiserum of B. thuringiensis ssp. kurstaki . The plasmid and protein profiles of B. thuringiensis STB-1 were compared with those of its reference strains, ssp. kurstaki and ssp. kenyae . To verifiy the gene type of B. thuringiensis STB-1, PCR analysis was performedwith Spodoptera -specific cry gene primers. The result showed that B. thuringiensis STB-1, unlike its reference strains, had cry1Aa , cry1Ab , cry1Ac and cry1E , suggesting that B. thuringiensis STB-1 was a unique strain with respect to gene type. In addition, B. thuringiensis STB-1 showed a high level of toxicity against both S. exigua and Bombyx mori , whereas B. thuringiensis ssp. kurstaki HD-1 or ssp. kenyae showed a high level of toxicity against only Bombyx mori or S. exigua , respectively.     

对鳞翅目害虫高毒力的Bt cry1Aa基因的分离克隆及表达

Bt菌Ly30株是我国自行分离的对多种害虫具有高毒力的苏云金芽孢杆菌,经CAPS(cleaved amplified polymorphic sequences)系统鉴定,它含有cry1Aa基因。以全长基因PCR产物的粘端定向克隆的方法, 设计一对特异引物,分别引入NcoⅠ和BamHⅠ/NcoⅠ酶切位点。以Ly30质粒DNA为模板扩增cry1Aa全长基因,与表达载体Pkk233-2相应酶切产物连接,转化大肠杆菌,获得含有cry1Aa基因重组质粒pKKLy1Aa。完成了该基因的亚克隆和序列测定,结果表明,该基因的编码区为3 531 bp,编码蛋白分子量为133.2kD,含1.176个氨基酸,等电点Pi为4.99。该基因序列已在GenBank中登记注册,登录号为AF384211,并被国际Bt杀虫晶体蛋白基因命名委员会正式命名为cry1Aa12。对重组菌KKLy1Aa进行诱导表达研究。在0.6 mmol/L IPTG、37℃、8 h培养条件下,该基因获得高效表达,SDS-PAGE电泳检测到明显的133.2 kD蛋白带。室内生测结果表明,Cry1Aa蛋白对不同的小菜蛾品系均有较高的杀虫活性,其LC₅₀值分别为0.203 μg/mL和0.554 μg/mL。     

Novel cry gene from Paenibacillus lentimorbus strain Semadara inhibits ingestion and promotes insecticidal activity in Anomala cuprea larvae

A positive clone was selected from a library of total cell DNA of Paenibacillus lentimorbus strain Semadara that reacted with an antiserum that was raised against parasporal crystal proteins produced by this strain. The positive clone had a DNA insert containing two whole cry genes (cry43Aa1, cry43Ba1), one partial cry gene (cry43-like), and three smaller genes located upstream. Eight blocks that are conserved in the Cry proteins of Bacillus thuringiensis [Microbiol. Mol. Biol. Rev. 62 (1998) 775] were detected in their deduced amino acid sequences. The Escherichia coli transformant expressing cry43Aa1 caused inhibition of ingestion and 90% mortality in the first stadium larvae of Anomala cuprea. A low concentration of sporangia mixed with the transformant expressing cry43Aa1 easily infected the larvae of A. cuprea. The protein of approximately 150 kDa produced by the transformants expressing the cry genes reacted with antiserum specific for the parasporal crystal proteins. Southern hybridization analysis demonstrated that the cry genes were located on the chromosomal DNA of this strain, which possessed at least four cry genes.