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。     

苏云金芽胞杆菌幕虫亚种晶胞粘连现象与晶体蛋白基因cry26Aa所在质粒有关

苏云金芽胞杆菌幕虫亚种T02菌株的伴胞晶体在芽胞外壁内侧形成,呈现晶胞粘连的现象。在此菌株中克隆了cry26 Aa和cry28 Aa两个基因,并对晶胞粘连现象与质粒的相关性做了系统研究。通过消除幕虫亚种T02菌株的质粒,得到了仅消除cry26 Aa所在质粒的菌株BMB1151和无质粒的菌株BMB1152。通过穿梭载体将cry26 Aa和cry28 Aa两个基因分别和同时转化无质粒突变株BMB1152并表达,形成的晶体与芽胞独立存在不能粘连,表明在幕虫亚种染色体背景下仅仅cry的表达不能形成晶胞粘连现象,从而推断晶胞粘连现象可能与幕虫亚种两个基因所在的质粒有关;进一步的研究发现将cry26 Aa在仅消除cry26 Aa所在质粒的突变株BMB1151中表达,形成的晶体与芽胞也分别独立存在不能粘连,从而进一步推断幕虫亚种晶胞粘连现象与cry26 Aa所在质粒有关。     

苏云金芽胞杆菌杀线虫伴胞晶体蛋白基因cry6Aa的克隆与表达

通过对晶体蛋白N-末端氨基酸测序,设计简并探针,从对根结线虫高毒力苏云金芽胞杆菌YBT-1518菌株中克隆到1个含有杀线虫晶体蛋白基因的片段。序列测定表明该序列含有两个ORF(orf1和orf2),其中orf1与基因cry6Aa1同源性为98%,已在GenBank上登录(Acc.NO.AF499736),并被命名为cry6Aa2。将克隆的该片段克隆到穿梭载体pHT304上,并转化苏云金芽胞杆菌无晶体突变株BMB171,重组菌株可形成米粒状伴胞晶体。生物测定表明,表达的毒素蛋白对北方根结线虫的LC50为9.47μg/mL,毒力与出发菌株(10.74μg/mL)相当。     

四川盆地生态区苏云金芽胞杆菌cry基因的鉴定及新型模式cry基因的克隆

[目的]为了明确四川盆地生态区土壤中苏云金芽胞杆菌cry,基因资源情况,进一步克隆出新型的杀虫晶体蛋白基因.[方法]本研究主要通过菌株晶体形状的光学显微镜及扫描电镜观察、PCR-RFLP技术鉴定cry基因型法、杀虫晶体蛋白的SDS-PAGE分析和菌株生物活性测定等方法对此地区菌株进行研究.[结果]从四川盆地不同生态区采集2650份土壤样品中分离了791株苏云金芽胞杆菌.PCR-RFLP鉴定结果表明:此地区的苏云金芽胞杆菌主要含有cry1,cry2,cry3,cry4/10,cry9,cry30和cry40等7种cry,基因类型;含cry1基因的菌株最丰富,共有21种不同cry1型基因组合;从中发现了新型模式基因,并采用Tail-PCR技术获得了其中3个基因的全长序列,被国际苏云金芽胞杆菌杀虫晶体蛋白基因命名委员会命名为cry54Aa1、cry30Fa1和cry30Ga1.通过生物活性测定,发现对鳞翅目和双翅目害虫具毒力的菌株.未鉴定出基因型的80个菌株的伴胞晶体SDS-PAGE分析表明:这些菌株均有40～130 kDa蛋白表达,极有可能含新型的杀虫蛋白基因.[结论]研究结果充分体现了四川盆地生态区苏云金芽胞杆菌资源的多样性及特殊性,所蕴含的杀虫蛋白基因在农业生产上具有重要意义和应用前景.     

苏云金芽胞杆菌sigK 基因插入失活突变体的特点及其对cry3A基因启动子活性的影响

摘要: 【目的】构建苏云金芽胞杆菌(Bacillus thuringiensis,简称Bt) sigK 基因插入失活突变体,分析突变体特点并明确其对cry3A 基因启动子的影响。【方法】采用同源重组技术在苏云金芽胞杆菌HD-73 菌株sigK 基因中插入卡那霉素抗性基因,构建了sigK 基因插入失活突变体。通过生长曲线测定、扫描电子显微镜观察晶体、芽胞形成情况和芽胞计数及SDS-PAGE 等方法分析了突变体的特点; 构建了遗传恢复菌株对上述性状进行了功能验证; 利用启动子融合lacZ 技术检测了cry3A 基因启动子的转录活性。【结果】获得了苏云金芽胞杆菌HD-73 菌株sigK 基因突变体,生长曲线测定表明,突变体较出发菌株在稳定期后期生长较慢; 扫描电子显微镜观察和芽胞计数分析显示,突变体丧失了形成芽胞和晶体的能力; SDS-PAGE 分析表明突变体中伴胞晶体蛋白的表达量明显低于出发菌株和恢复菌株。利用载体pHT315 携带sigK 基因及其启动子在突变株中表达,所获得的遗传恢复菌株恢复了突变株产生芽胞和晶体的能力; sigK 基因的突变可以提高cry3A 基因启动子在产胞后期的转录活性,对cry3A 启动子指导的Cry 蛋白表达量没有显著影响。【结论】本研究证明sigK 基因为苏云金芽胞杆菌芽胞形成所必需,并影响伴胞晶体蛋白的产量; sigK 基因功能的丧失有利于cry3A 基因启动子在产胞后期的转录。     

带cry3Aa启动子的aiiA基因在苏云金芽胞杆菌中的表达

N 乙酰高丝氨酸内酯 (N acyl homoserinelactones,AHLs) ,是一类数量感知 (Quorum sensing)系统中的信号分子 ,它参与诱导调控许多植物病原菌致病基因的表达。苏云金芽胞杆菌的AiiA蛋白能降解这类AHLs分子 ,进而可减弱病原菌致病基因表达产生的病害。苏云金芽胞杆菌杀虫晶体蛋白基因cry3Aa的启动子是一种不依赖芽胞形成的启动子 ,它相对于其它cry类基因的启动子有启动基因转录时间早 ,转录时间长的优点。通过重叠延伸PCR ,用杀虫晶体蛋白基因cry3Aa启动子替换编码AiiA蛋白的基因aiiA自身的启动子 ,构建了融合基因pro3A aiiA。将融合基因装入穿梭载体pHT3 0 4的BamHI SphI位点 ,得到重组质粒pBMB686并转化苏云金芽胞杆菌无晶体突变株BMB171,重组菌株BMB686的AiiA蛋白表达量在各个生长时期均高于对照菌株 ,对AHLs分子的降解活性和对胡萝卜软腐欧文氏菌感染马铃薯产生病害的抑制能力也明显优于对照菌株     

苏云金芽胞杆菌G03 spoIVF操纵子敲除对芽胞和晶体形成的影响

spoIVF是一个普遍存在于芽胞杆菌中的操纵子。在枯草芽胞杆菌中,它编码的两个蛋白是芽胞形成所必需的。采用基因重组技术敲除了苏云金芽胞杆菌G03菌株中的spoIVF操纵子,构建了spoIVF缺失株G03(spoIVF-)。研究表明:该突变株丧失了形成芽胞和晶体的能力。lacZ基因与cry1Aa基因的启动子融合表达分析发现:突变株中的cry1Aa基因的活性严重降低。利用载体pSTK携带spoIVF操纵子在突变株中的表达,使突变株部分恢复了产胞和形成杀虫晶体蛋白的能力。这说明spoIVF操纵子是所必需的,同时该操纵子还影响σE因子控制的cry1Aa基因表达。     

类似S-层蛋白的苏云金芽胞杆菌伴胞晶体蛋白基因的克隆

芽胞杆菌CTC菌株被鉴定为苏云金芽胞杆菌 ,鞭毛血清型H2 ,幕虫亚种 ;产生卵圆形伴胞晶体 ,伴胞晶体蛋白为 1 0 0kD ;测定了该蛋白的N 末端序列 ,该序列与炭疽芽胞杆菌的细胞表面S 层蛋白具 92 %～ 93%相似性 ;根据Southern杂交制作了该晶体蛋白基因ctc所在位置的限制性酶切图谱 ,分别克隆了该基因 5′和 3′端所在的 2 9kbXbaI片段和 3 1kbClaIDNA片段 ,彼此间具 0 6kb重叠 ,通过拼接获得含完整ctc基因的克隆。含该基因的大肠杆菌与表达S 层蛋白的大肠杆菌具相似生长特征。初步表明CTC菌株的伴胞晶体由细胞表面S 层蛋白组成。苏云金芽胞杆菌区别于蜡状芽胞杆菌和炭疽芽胞杆菌的唯一标准是能形成伴胞晶体 ,由于S 层是细胞表面的结构成分 ,本文对CTC菌株鉴定为苏云金芽胞杆菌以及伴胞晶体作为苏云金芽胞杆菌鉴别的唯一标准提出了质疑     

苏云金芽胞杆菌大质粒pBMB28的克隆

苏云金芽胞杆菌幕虫亚种YBT-020具有典型的晶胞粘连表型。在前期的研究中,通过质粒消除实验,推测晶胞粘连现象与YBT-020内生质粒pBMB28有关。为了定位质粒pBMB28上控制晶胞粘连表型的基因,首先对质粒pBMB28进行克隆。利用穿梭载体pEMB0557,成功构建了苏云金芽胞杆菌YBT-020的基因组人工染色体(BAC)文库。前期的研究表明晶体蛋白基因cry28Aa定位在质粒pBMB28上,根据cry28Aa基因序列设计引物,从文库中筛选到含有cry28Aa的重组质粒pBMB231。镜检和SDS-PAGE证明质粒pBMB231转化无晶体突变株BMB171形成的重组子BMB231可以产生Cry28Aa晶体蛋白,但不能恢复晶胞粘连表型。对重组质粒pBMB231的插入片段末端序列测定并设计引物筛选文库,通过染色体步移方式得到4个可以重叠覆盖质粒pBMB28不同区域的克隆子,从而克隆了该质粒。对这4个克隆子末端测序和酶切分析,测算出该质粒的大小约为140 kb。进一步确定应用基因组BAC文库以及重叠片段筛选的方法,可以快速有效的克隆苏云金芽胞杆菌大质粒。     

类似S-层蛋白的苏云金芽胞杆菌伴胞晶体蛋白基因的克隆

芽胞杆菌CTC菌被鉴定为苏云金芽胞杆菌,鞭毛血清型H2,幕虫亚种;产生卵圆形伴胞晶体,伴胞晶体蛋白为100kD;测定了该蛋白 N－末端序列,该序列与炭疽芽胞杆菌的细胞表面S－层蛋白具92－93％相似性,根据Southern杂交制作了该晶体蛋白基因ctc所在位置的限制性酶切图谱,分别克隆了该基因5’和3’端所在2．9kb XbaI片段和3．1kb Cla I DNA片段,彼此间具0．6kb重叠,通过拼接获得含完整ctc基因的克隆,含该基因的大肠杆菌与表达S－层蛋白的大肠杆菌具相似生长特征,初步表明CTC菌株的伴胞晶体由细胞表面S－层蛋白组成,苏云金芽胞杆菌区别于蜡状芽胞杆菌和炭疽芽胞杆菌的唯一标准是能形成伴胞晶体,由于S－层是细胞表面的结构成分,本文对CTC菌株鉴定为苏云金芽胞杆菌以及伴胞晶体作为苏云金芽胞杆菌鉴别的唯一标准提出了质疑。     

苏云金芽孢杆菌Rpp02菌株cry1Ac20基因的克隆及表达

Rpp02菌株是本实验室分离的一株对鳞翅目等多种害虫具有高毒力的苏云金芽孢杆菌莫里逊亚种 (Bacillus thuringiensis subsp. morrisoni), 经PCR检测,它含有cry1Ac基因。对其基因组DNA进行PCR扩增,得到大约4 kb的产物。测序结果表明,该片段含有一个较大的ORF框,基因编码区为3 534 bp,编码1 177个氨基酸,分子量为133.144 kD,pI 4.952, 为弱酸性蛋白质,亮氨酸（Leu）、丝氨酸（Ser）、谷氨酸（Glu）3种氨基酸含量最高,分别为8.0%、7.8%、7.7%。该基因序列与cry1Ac序列同源性达到99%,并被国际Bt杀虫晶体蛋白基因命名委员会命名为cry1Ac20。生物测定表明,该基因在大肠杆菌中得到了表达,表达产物具有较强的杀虫效果,试喂菜青虫48 h后,校正死亡率为88.78%。     

Cloning and expression of two crystal protein genes, cry30Ba1 and cry44Aa1, obtained from a highly mosquitocidal strain, Bacillus thuringiensis subsp. entomocidus INA288

Two novel crystal protein genes, cry30Ba and cry44Aa, were cloned from Bacillus thuringiensis subsp. entomocidus INA288 and expressed in an acrystalliferous strain. Cry44Aa crystals were highly toxic to second-instar Culex pipiens pallens (50% mortality concentration [LC50] = 6 ng/ml) and Aedes aegypti (LC50 = 12 ng/ml); however, Cry30Ba crystals were not toxic.     

Cloning and study of the expression of a novel cry1Ia-type gene from Bacillus thuringiensis subsp. kurstaki

AIMS: Cloning and expression of a new cry1Ia-type gene of Bacillus thuringiensis. METHODS AND RESULTS: PCR amplification, using gene cry1I-specific primers revealed the presence of such a gene in the strain BNS3 of Bacillus thuringiensis subsp. kurstaki. The cloning and sequencing from BNS3 of the cry1Ia-type gene, called crybns3-3, showed an open reading frame of 2160-bp, encoding a protein of 719 amino acid residues. Both nucleotide and amino acid sequences similarity analysis revealed that the crybns3-3 is a new cry1Ia-type gene, presenting several differences from the cry1Ia-type genes. The study of the expression of crybns3-3 by Northern blot and RT-PCR showed that it was transcribed. The expression of crybns3-3 under the control of BtI and BtII promoters revealed that Crybns3-3 would co-crystallize with the endogenous delta-endotoxins. CONCLUSIONS: crybns3-3 is a novel cry1Ia gene isolated from B. thuringiensis subsp. kurstaki strain BNS3. SIGNIFICANCE AND IMPACT OF THE STUDY: The characteristics of crybns3-3 indicate that it is a new cry1Ia-type gene. Amino acid residue substitutions presented in Crybns3-3 could be exploited for both toxicity and specificity studies. Crybns3-3 would interact and co-crystallize at least partially with the endogenous delta-endotoxins of BNS3, and then participate in the formation of the parasporal crystal inclusions.     

Broadening the insecticidal spectrum of Lepidoptera-specific Bacillus thuringiensis strains by chromosomal integration of cry3A

A TnpI-TnpIA-mediated and thermosensitive recombination system was developed to construct genetically modified Bacillus thuringiensis strains encoding a crystal protein particularly active against Coleopteran species. Based on B. thuringiensis transposon Tn4430, an integrative vector, pBMB-R14E, was constructed, by which the cry3A delta-endotoxin gene highly toxic to Lepidoptera was delivered into a wildtype B. thuringiensis subsp. kurstaki strain YBT1520. The cry3A gene was integrated into the chromosome of the host strain. Then the integrative vector was eliminated by moving recombinant cultures to 46 degrees C. Two recombinant B. thuringiensis strains, BMB1520-S and BMB1520-T, were obtained. In recombinant strains, the cry3A gene was stably expressed in measurable amounts and did not reduce the expression of endogenous crystal protein genes. Bioassay results showed that BMB1520-S and BMB1520-T, in addition to the activity against lepidopteran Plutella xylostella third-instar larvae present in the parental strains, exhibited a high level of activity against coleopteran Rhyllodecta vulgatissima third-instar larvae, absent from the parental strains.     

Gene clusters located on two large plasmids determine spore crystal association (SCA) in Bacillus thuringiensis subsp. finitimus strain YBT-020

Crystals in Bacillus thuringiensis are usually formed in the mother cell compartment during sporulation and are separated from the spores after mother cell lysis. In a few strains, crystals are produced inside the exosporium and are associated with the spores after sporulation. This special phenotype, named 'spore crystal association' (SCA), typically occurs in B. thuringiensis subsp. finitimus. Our aim was to identify genes determining the SCA phenotype in B. thuringiensis subsp. finitimus strain YBT-020. Plasmid conjugation experiments indicated that the SCA phenotype in this strain was tightly linked with two large plasmids (pBMB26 and pBMB28). A shuttle bacterial artificial chromosome (BAC) library of strain YBT-020 was constructed. Six fragments from BAC clones were screened from this library and discovered to cover the full length of pBMB26; four others were found to cover pBMB28. Using fragment complementation testing, two fragments, each of approximately 35 kb and located on pBMB26 and pBMB28, were observed to recover the SCA phenotype in an acrystalliferous mutant, B. thuringiensis strain BMB171. Furthermore, deletion analysis indicated that the crystal protein gene cry26Aa from pBMB26, along with five genes from pBMB28, were indispensable to the SCA phenotype. Gene disruption and frame-shift mutation analyses revealed that two of the five genes from pBMB28, which showed low similarity to crystal proteins, determined the location of crystals inside the exosporium. Gene disruption revealed that the three remaining genes, similar to spore germination genes, contributed to the stability of the SCA phenotype in strain YBT-020. Our results thus identified the genes determining the SCA phenotype in B. thuringiensis subsp. finitimus.     

Two novel delta-endotoxin gene families cry26 and cry28 from Bacillus thuringiensis ssp. finitimus.

Genes cry26Aal and cry28Aal were cloned from Bacillus thuringiensis ssp. finitimus strain B-1166 VKPM. This strain forms insecticidal crystal bodies either outside or inside the exosporium. The deduced amino acid sequence of the cry26Aal gene product included seven residues determined to be an N-terminal part of a chymotrypsin-treated delta-endotoxin isolated from the same strain. Earlier this protein was detected in both free and spore-associated types of crystals [Revina et al., Biokhimia (1999) in press]. Neither BtI nor BtII promoter sequences were found upstream of the open reading frames in both genes. Southern hybridization has shown that the surroundings of both genes at least 3 kb upstream and downstream of the open reading frames are unique. We suggest that the protein Cry26Aal in both types of crystal bodies is synthesized under the control of one and the same genomic locus.     

Comparative study on effect of different promoters on expression of cry1Ac in Bacillus thuringiensis chromosome

AIMS: The aim of this work was to investigate the effect of cry3A promoter on the expression of cry1Ac in Bacillus thuringiensis chromosome and stably enhance the production of different cry genes under the control of cry3A promoter. METHODS AND RESULTS: The cry1Ac gene, which is specific to Lepidopteran larvae, was integrated into the chromosome of a B. thuringiensis plasmid-free and acrystalliferous strain BMB171, under the control of cry3A promoter and cry1Ac promoter, respectively. The expression of cry1Ac genes in the chromosome of host strain was investigated. The results from sodium dodecyl sulfate-polyacrymide gel electrophoresis, crystal observation and bioassay showed that either integrated with cry3A promoter (cry3Apro-cry1Ac) or with its native promoter (cry1Acpro-cry1Ac), cry1Ac gene could efficiently and stably express in the chromosome. The production of cry3Apro-cry1Ac gene was higher than that of cry1Acpro-cry1Ac gene. CONCLUSIONS: The cry3A promoter enhanced the expression of cry1Ac gene efficiently either on the chromosome or on the plasmid in B. thuringiensis strain. SIGNIFICANCE AND IMPACT OF THE STUDY: So far, the comparative studies on cry3A promoter and other cry promoters were carried on B. thuringiensis plasmids. This system offers an additional method for potentially improving the efficacy of B. thuringiensis insecticidal proteins efficiently, stably and safely.     

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。     

Conjugative transfer, stability and expression of a plasmid encoding a cry1Ac gene in Bacillus cereus group strains

The plasmid pHT73 containing cry1Ac and tagged with an erythromycin resistance gene was transferred from Bacillus thuringiensis subspecies kurstaki KT0 to several Bacillus cereus group strains by conjugation. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and phase contrast microscopy showed that the transconjugants containing plasmid pHT73 could express Cry1Ac toxin and produce bipyramidal crystalline inclusion bodies during sporulation. The study demonstrated that pHT73 could be transferred to B. thuringiensis subsp. kurstaki, several B. cereus strains and Bacillus mycoides. Under non-selective conditions, the stability of the pHT73 plasmid in the transconjugants was found to be 58.2-100% after 100 generations and 4-96% after 200 generations. The variations are mainly caused by the choice of receptor strain.