转化cry1C基因对苏云金芽胞杆菌杀虫活性的影响

将含基因cry1C的质粒,通过电脉冲法转入含基因cry1C的质粒,通过电泳冲法转入含基因cry1Ab、cry1Ac和cry2的对小菜蛾具有高毒力的苏云金芽胞杆菌野生菌株YBT－803－1中,得到转化子MBMY－003。PCR和SDS－PAGE分析显示,cry1C可在其中正常复制、表达,但使受体菌部分内源质粒发生丢失。生物测定结果表明,转化子MBMY－003既对甜菜蛾有毒力,LC50值为1．178μL／mL,高于出发菌株YBT－803－1（LC501．879μL／mL）,也对小菜蛾有毒力,LC50值为1．968μL／mL,低于YBKT－803－1（LC501．143μL／mL）。表明cry1C转入后,提高了野生菌株YBT－803－1对甜菜夜蛾的毒力,却降低了对小菜蛾的毒力。     

苏云金芽胞杆菌cryⅡ基因的克隆和表达

分离的苏云金芽胞杆菌(Bacillus-thuringiensis)YBT-791对鳞翅目小菜蛾(Plutellaxylostella)有毒力,将其质粒DNA提纯,经HindⅢ酶切后与杀虫晶体蛋白cryⅡ基因探针杂交,显示出分子量分别为5kb和9．4kb两条DNA阳性片段。把5kb的DNA阳性片段克隆到pUCl8的HindⅢ位点上并转化大肠杆菌TGl,经酶切和杂交检测,证明斑点杂交阳性克隆子中含有5kb的cryI片段。把这个含cryⅡ基因的5kbHindⅢ片段进行亚克隆,将4kb的BamHI－Pstl酶切片段插入穿梭载体pXl61中,并用电脉冲法克隆于苏云金杆菌不产伴胞晶体的突变株中,得到产生单一cry Ⅱ基因编码的杀虫晶体蛋白的克隆菌株M一5。经电镜观察,该克隆菌株能形成出发菌YBT－791多种形态伴胞晶体中的一种方形伴胞晶体;经免疫双扩试验,它只能与Cry Ⅱ晶体蛋白抗血清形成沉淀线;经SDS—PAGE电泳,克隆菌的伴胞晶体只含有一种65kDa的晶体蛋白。生物测定结果表明它既对鳞翅目小菜蛾(Piutella xylostella)有毒性,又对双翅目致倦库蚊(Culex quinquefasciatus)有毒性。     

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）ＴＢＴ－１５２０菌株含有两个杀虫晶体蛋白基因片段,其５’＝末端所在ＨｉｎｄⅢ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ｃｒｙ１Ａｃ基因     

苏云金芽胞杆菌cry2Ab、cry9Ea基因的表达及活性分析

旨在为农业害虫防治提供更多的苏云金芽胞杆菌基因资源,从中国吉林市龙潭山土壤样品中分离得到野生菌株命名为Bt LTS-7,扫描电镜显示该菌株产生晶体形状为双锥体和正方体,聚丙烯酰胺凝胶电泳显示该菌株产生130 kD和71 kD的晶体蛋白,通过PCR鉴定出该菌株中含有cry2Ab和cry9Ea基因,并成功克隆到了这两个新基因,并被Bt国际命名委员会正式命名为cry2Ab28和cry9Ea9,将两个基因分别在大肠杆菌Rosetta( DE3)中表达,并进一步对其表达蛋白进行杀虫活性测定.结果显示,Cry2Ab28蛋白对棉铃虫(Helicoverpa armigera)初孵幼虫具有杀虫活性,LC50为32.45 μg/mL.Cry9Ea9蛋白对小菜蛾初孵幼虫(Plutella xylostella)具有较高杀虫活性,LC50为0.77μg/mL.     

苏云金芽胞杆菌LM1212菌株cry基因启动子的活性分析

【背景】前期发现一株具有独特分化表型的苏云金芽胞杆菌(Bacillusthuringiensis,Bt)LM1212,该菌株共有14种杀虫基因,组成了10个转录单元。利用源自菌株LM1212的晶体产生细胞调控因子(crystal producing cell regulator, CpcR)以及其可以激活的cry35-like基因启动子,已在典型Bt菌株HD73中成功构建了非芽胞杀虫蛋白表达体系。【目的】比较菌株LM1212的不同杀虫基因启动子的转录活性,明确可被转录因子CpcR激活的且转录活性较高的启动子,以此为基础优化非芽胞杀虫蛋白表达体系。【方法】将10个启动子区域分别与lacZ报告基因融合构建在pHT304-18Z载体上,得到了10个重组质粒;然后将cpcR基因及其启动子(PcpcR-cpcR)分别反向构建在选取的启动子区域与lacZ报告基因的上游,得到可以表达CpcR且与上述构建相对应的10个重组质粒,将这些重组质粒分别转入不含CpcR的菌株HD73,即获得20个可用于测定β-半乳糖苷酶活性的重组菌株。通过光学显微镜观察和SDS-PAGE检测明确杀虫蛋白表达的情况。【结果】在...     

苏云金芽胞杆菌杀虫晶体蛋白基因的分类

１９８９年Ｈｏｆｔｅ和Ｗｈｉｔｅｌｅｙ根据氨基到序列的同源性和杀虫谱双重标准将苏云金芽胸杆菌杀虫晶体蛋白基因划分为５类１４亚类。根据双重标准出现的问题和冲突,给分类带来了困扰,为此１９９５年,只根据氨基酸序列的同源性进行分类。现已将基因分为２１群,４４亚群,６４类,１１６个亚类。本文介绍了纳入新系统的条件、分类原则、命名方法、定名步骤和分类中值得注意的问题。     

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cry2Ad基因的克隆及其表达产物的活性分析

苏云金芽胞杆菌(Bacillus thuringiensis,Bt)SBT2是我国新分离出的一株野生菌株.扫描电镜显示该菌株产生双锥体形晶体.琼脂糖凝胶电泳发现其质粒图谱含有5个条带.聚丙烯酰胺凝胶电泳显示此菌株产生130 kD晶体蛋白.利用PCR-RFLP法进行杀虫基因类型鉴定,发现其含有cry1Aa、cry1Da、cry1Hb、cry1Jb、cry1Ka 、cry1Ib、基因.Cry2Ad蛋白的活性至今未见研究报道,本研究克隆和测序了该基因.并对其进行了表达.生物活性测定结果表明其表达产物对舞毒蛾(Lymantria dispar)、棉铃虫(Helicoverpa armigera)、亚洲玉米螟(Ostrinia furnacalis)、小菜蛾(Plutella xylostella)有低活性;对大猿叶甲(Colaphellus bowringi)无活性.     

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。     

Rapid cloning, identification, and application of one novel crystal protein gene cry30Fa1 from Bacillus thuringiensis

In this study, a fast and efficient strategy has been developed for identifying and isolating novel cry genes from Bacillus thuringiensis by combining the PCR-restriction fragment length polymorphism and the single-oligonucleotide nested-PCR method. Using this method, one novel holotype cry gene, cry30Fa1 , encoding a polypeptide of 687 amino acid residues with a molecular mass of 77.1 kDa, 74% identical to Cry30Aa1, was cloned from the B. thuringiensis strain BtMC28. Furthermore, the cry30Fa1 gene was successfully expressed in Escherichia coli BL21 (DE3). The Cry30Fa1 proteins, isolated from the cultures of recombinant E. coli , had remarkable insecticidal effects against Plutella xylostella and Aedes aegypti with LC50 at 6.477 and 15.359 μg mL⁻¹, respectively. Our results strongly suggest that this strategy is highly efficient and advantageous in terms of rapid cloning of holotype cry genes that have minimal identity to known genes. The cloning of the cry30Fa1 gene would be useful in the resources of the insecticidal crystal genes and may serve as an alternative choice of an insecticide for potential problems associated with insect resistance.     

Characterization and cloning of the cry2A gene from indigenous isolates of Bacillus thuringiensis

Molecular Biology - Discovery of novel cry genes of Bacillus thuringiensis (Bt) with higher toxicity is important for the development of transgenic Bt crops resistant to target pests. Two new...     

广西大王岭和大明山自然保护区苏云金芽孢杆菌收集与鉴定

从广西大王岭和大明山两个自然保护区共采集到土样264份,共分离出597株芽孢杆菌,通过光学和电子显微镜检观察,16株分离株观察到伴胞晶体蛋白,初步确定为苏云金芽胞杆菌(Bacillus thuringiensis,简称Bt),出菌率为6.06%.在16株Bt分离株中,有4株在芽孢形成过程中能产菱形晶体蛋白,其余12株能产圆形和其他形状的晶体蛋白.利用PCR-RFLP方法和SDS-PAGE方法对16株Bt分离菌进行了蛋白和基因型的鉴定,结果表明,16株分离株中含有4株cry1Ac基因,表达约130 kD的晶体蛋白,其中含有cry30基因和cry40基因的菌株分别1株和3株,表达大约75 kD的晶体蛋白;另外8株Bt菌株表达蛋白大小不一,其基因型尚不能确定,有待进一步分析.生物测定表明,产菱形晶体含有cry1Ac基因的4株Bt分离株对鳞翅目小菜夜蛾幼虫有很强的毒杀活性,而其它分离株对小菜夜蛾没有毒杀活性.     

黑龙江凉水自然保护区苏云金芽孢杆菌的收集与鉴定

估,实验结果表明东北森林地区具有丰富多样的Bt菌株和杀虫基因资源.     

Characterization of a novel cry2Aa-type gene from Bacillus thuringiensis subsp. kurstaki

A 4 kb BamHI-HindIII fragment, corresponding to the cry2A operon of Bacillus thuringiensis subsp. kurstaki strain BNS3, was cloned. The sequencing of the corresponding cry2Aa-type gene, termed crybns3-4, revealed an open reading frame of 1902 bp, encoding a protein of 633 amino-acid residues. Both nucleotide and amino-acid sequences similarity analysis revealed that crybns3-4 is a new cry2Aa-type gene which has several differences from the reported cry2Aa-type genes. The transfer of the cloned operon to an acrystalliferous mutant of BNS3, revealed an expression of the new cry2Aa-type gene and a production of parasporal crystal inclusions in the transformants.     

Characterization of one novel cry8 gene from Bacillus thuringiensis strain Q52-7

Bacillus thuringiensis (Bt) is the most widely used insecticidal microbe due to its specific toxicity and safe use with respect to animals and the environment. In this study, we isolated Bt strain Q52-7 from a soil sample collected in the Qian Shan District, Liao Ning Province, China. We observed that the Q52-7 strain produced spherical crystals. The Bt Q52-7 strain had high toxicity against Asian Cockchafer (Holotrichia parallela), exhibiting an LC₅₀ of 3.80 × 10⁹ cfu/g, but is not toxic for Anomala corpulenta Motschulsky and Holotrichia oblita. Using general cry8 primers, we amplified a 1.3 kb fragment with the polymerase chain reaction. Specific primers were designed for the amplified fragment to clone the full-length coding region. A novel gene, cry8Na1, had 69 % sequence similarity with cry8Ca1. cry8Na1 gene was successfully expressed in the HD-73^– acrystalliferous mutant of Bt subsp. Kurstaki HD-73. Bioassays demonstrated that the Cry8Na1 protein is highly toxic for the H. parallela, with a 50 % lethal concentration of 8.18 × 10¹⁰ colony forming units per gram.     

Cloning of a novel crystal protein gene cry 1K from Bacillus thuringiensis subsp. morrisoni

Abstract In Escherichia coli with group II capsules, the synthesis of capsular polysaccharide and its cellular expression are encoded by the kps gene cluster, which is composed of three regions. The central region 2 encodes proteins involved in polysaccharide synthesis, and the flanking regions 1 and 3 direct the translocation of the finished polysaccharide across the cytoplasmic membrane and its surface expression. The kps genes of E. coli with the group II capsular K5 polysaccharide, have been cloned and sequenced. Region 1 contains the kps E, D, U, C and S genes. In this communication we describe the overexpression of the kps D and kps U genes as well as the isolation of the KpsU protein from the recombinant bacteria by chloroform treatment. The purified KpsU protein exhibited CMP-Kdo-synthetase activity. The N-terminal sequence and two internal peptide sequences of the isolated protein are in agreement with that previously predicted from the DNA sequence of the kps U gene. The kinetic data of the CMP-Kdo-synthetase participating in K5 capsule expression (K-CMP-Kdo-synthetase) differ from those described for the CMP-Kdo-synthetase, participating in lipopolysaccharide synthesis (L-CMP-Kdo-synthetase).     

Cloning and nucleotide sequence of a novel cry gene from Bacillus thuringiensis

A cry1Ab-type gene was cloned from a new isolate of Bacillus thuringiensis by PCR. When restriction pattern was compared with that of known genes it was found to have additional restriction site for ClaI. Nucleotide sequencing and homology search revealed that the toxin shared 95% homology with the known Cry1Ab proteins as compared to more than 98% homology among the other reported Cry1Ab toxins. The gene encoded a sequence of 1,177 amino acids compared to 1,155 amino acids encoded by all the other 16 cry1Ab genes reported so far. An additional stretch of 22 amino acids after the amino acid G793 in the new toxin sequence showed 100% homology with several other cry genes within cry1 family. Homology search indicated that the new cry1Ab-type gene might have resulted by nucleotide rearrangement between cry1Ab and cry1Aa/cry1Ac genes.     

Characterization of Vegetative Insecticidal Protein vip Genes of Bacillus thuringiensis from Sichuan Basin in China

Vegetative insecticidal proteins (Vip), the second generation of insecticides, are produced during the vegetative growth stage of Bacillus thuringiensis (Bt). To perform a systematic study of vip genes in Bt strains from different ecological regions of Sichuan Basin, 1,789 soil samples were collected from this basin, which is situated in the western region of China. The basin has a complicated geomorphology and contains mountains, forests, highlands, hursts, and plains. A total of 2,134 Bt strains have been screened from the 1,789 soil samples. According to the results, three vip-type genes were found in this basin, namely the vip1, vip2, and vip3-type genes. Strains containing vip3-type genes were the most abundant in our collection (67.4%), followed by vip2-type genes (14.6%) and vip1-type genes (8.1%). The three types of vip genes were distributed in most of the regions, but E Mei Mountain and the Ba Lang Mountains only contained vip3 genes in environments with high elevation, low temperature, insufficient oxygen, and abundant snow. Moreover, five novel vip3 genes were found, and these Vip proteins were toxic for Chilo suppressalis. All the results mentioned above suggest that Sichuan Basin is a rich resource for vip genes.     

Isolation and characterization of a novel insecticidal crystal protein gene from Bacillus thuringiensis subsp. aizawai.

Bacillus thuringiensis subsp. aizawai EG6346, a novel grain dust isolate, was analyzed by Southern blot hybridization for its insecticidal crystal protein (ICP) gene profile. Strain EG6346 lacks previously characterized cryIA ICP genes yet does possess novel cryI-related gene sequences. A recombinant genomic plasmid library was constructed for strain EG6346 in Escherichia coli. One recombinant plasmid, pEG640, isolated from the library contained a novel ICP gene on a 5.7-kb Sau3A insert. The sequence of this gene, designated cryIF, was related to, but distinct from, the published sequences for other cryI genes. A second novel cryI-related sequence was also located on pEG640, approximately 500 bp downstream from cryIF. Introduction of cryIF into a Cry- B. thuringiensis recipient strain via electroporation enabled sufficient production of CryIF protein for quantitative bioassay analyses of insecticidal specificity. The CryIF crystal protein was selectively toxic to a subset of lepidopteran insects tested, including the larvae of Ostrinia nubilalis and Spodoptera exigua.