苏芸金杆菌广谱杀虫Tml3—14菌株晶体毒素及毒力特性

以新筛选的对鞘翅目,鳞翊目、双翅目均具毒性的B.t.Tml3-14菌株为材料,用HD-1、3370-1为参考菌株,比较了伴孢晶体蛋白的多肽组成,以及经三种昆虫肠道酶降解产生的抗酶多肽组分。SDS-PAGE分析表明：Tml3-14晶体蛋白含1 38kD、l 32kD主要成分和65kD次要我分。其晶体分别由三种昆虫肠道酶消化产生的毒性多肽,经生物测定,杀家蚕的毒性成分为68.5kD、59kD多肽;杀斜纹夜蛾毒性成分为71kD,67kD和59.6kD多肽;杀马铃薯瓢虫毒性成分为69kD、65kD多肽。它与HD01、3370-1晶体均有明显差异。     

昆虫肠道蛋白酶作用下δ内毒素的毒性肽及毒力特异性的变化*

本文报道杀斜纹夜娥(Prodenia lirura)δ-内毒素和杀鞘翅目昆虫δ-内毒素经昆虫肠液蛋白酶及胰蛋白酶作用后,其毒性肽及毒力特异性的变化。 以Sephadex G75柱层析提纯的130kD原毒素,经斜纹夜蛾幼虫肠液蛋白酶作用后,产生的70kD与75kD抗蛋白酶多肽(PRP)对斜纹夜蛾和家蚕皆有毒;经家蚕幼虫肠液蛋白酶作用后,产生的62kD与65kD的PRP失去对斜纹夜蛾的毒性,仅对家蚕有毒;经胰蛋白酶作用后产生的65kD与68kD的PRP,其毒力特性与经家蚕肠液蛋白酶作用后的相似。斜纹夜蛾肠液蛋白酶作用后产生的PRP,可进一步被家蚕肠液蛋白酶或胰蛋白酶降解为63-65kD的PRP,此种多肽对斜纹夜蛾无毒,对家蚕有毒。杀鞘翅目昆虫的原毒素不能为胰蛋白酶和粘虫肠液所完全降解。证明不同种类昆虫的肠道蛋白酶对占-内毒素蛋白质的作用位点不同,δ-内毒素对宿主昆虫的毒力特异性与其肠道蛋白酶的特性密切相关。     

苏芸金杆菌广谱杀虫Tm13－14菌株晶体毒素及毒力特性

以新筛选的对鞘翅目、鳞翅目、双翅目均具毒性的Ｂ．ｔ．Ｔｍ１３－１４菌株为材料,用ＨＤ－１、３３７０－１为参考菌株,比较了伴孢晶体蛋白的多肽组成,以及经三种昆虫肠道酶降解产生的抗酶多肽组分。ＳＤＳ－ＰＡＧＥ分析表明：Ｔｍ１３－１４晶体蛋白含１３８ｋＤ、１３２ｋＤ主要成分和６５ｋＤ次要成分。其晶体分别由三种昆虫肠道酶消化产生绚毒性多肽,经生物测定,杀家蚕的毒性成分为６８．５ｋＤ、５９ｋＤ多肽;杀斜纹夜蛾毒性成分为７１ｋＤ,６７ｋＤ和５９．６ｋＤ多肽杀马铃薯瓢虫毒性成分为６９ｋＤ、６５ｋＤ多肽。它与ＨＤ－１、３３７０－１晶体均有明显差异。     

苏芸金杆菌伴孢晶体和芽孢孢衣的蛋白质组分及其与毒力的关系

研究了苏芸金杆菌(Bacillus thuringiensis)3个变种6个菌株的提纯伴孢晶体和芽孢对大蜡螟(Galleria mellonella)和大菜粉蝶(Pieris brassicae)的毒力、晶体的蛋白质和多肽成分及芽孢衣中的类晶体蛋白质成分.生物测定表明,晶体毒力高于芽孢,在总数量相同的情况下,晶体和芽孢近1:1的混合物的毒力高于单独的晶体或芽孢.芽孢衣中存在一种类似晶体蛋白质的成分,无晶体突变株及无效野生株的芽孢则无此种蛋白质且对两种昆虫无毒.变种wuhanensis和变种galleriae的晶体含MW138000的主要蛋白质和63000的次要蛋白质,经碱性缓冲液溶解后,上清液含MW138000的蛋白质,沉淀中含MW63000的蛋白质;变种aizawai的晶体中仅含138000的蛋白质.对大蜡螟无毒的HD-11(var.aizawai)晶体的蛋白质成分有别于上述晶体,其胰蛋白酶消化物的SDS凝胶电泳图型显示少2条多肽带,但对大菜粉蝶仍有效.结果表明,苏芸金杆菌的芽孢在昆虫病理中有重要作用,伴孢晶体的蛋白质和多肽成分与它们对昆虫的毒力特性之间有密切关系.     

土壤来源苏云金芽孢杆菌的形态、δ-内毒蛋白质及其毒力特性

在透射和扫描电镜下观察了土壤来源的94株苏云金芽孢杆菌(Bacillus thuringiensis)的菌体、鞭毛、芽孢及伴孢晶体的形态。以快速SDS—PAGE法分析了全部菌株δ-内毒素的蛋白质成分。用鳞翅目、鞘翅且及双翅目的9种昆虫进行了毒力试验。筛选出了数株杀鞘翅目和杀夜蛾科害虫的高效菌。发现了一些新型伴孢晶体,其形态和蛋白质成分均未报道过。     

乳化剂和保存条件对两种苏芸金杆菌杀蚊δ—内毒素的形响

我们已研究过有关化学物质及保存条件对苏芸金杆菌杀鳞翅目δ-内毒素的影响,本研究的目的在于考查乳化剂及保存温度与时间对两种杀蚊δ-内毒素毒力的影响。一、材料与方法 1、供试昆虫致倦库蚊(Culex fatigans)。 2、苏芸金杆菌菌株IPS—82(Bcillus thuringiensis var israelensis)L14(未鉴定)。 3、伴孢晶体的分离提纯以液体双相法结合蔗糖密度梯度法进行,提纯的晶体悬液于-20℃保存备用。     

杀鞘翅目苏云金芽孢杆菌新菌株及其杀虫剂的研究

从中国土壤中分离出2株杀鞘翅目昆虫的苏云金芽孢杆菌(Bacillus thuringiensis) YM03及SHQ11-10。YM03的血清型为H8a8b,SHQ1110的H血清型未知。二菌株皆产近菱形的薄扁伴孢晶体,分别含68～70kD和65kD的晶体蛋白质。毒力生物测定证明对柳蓝叶甲(Plagiodera versicolora)及马铃薯甲虫(Leptinotarsa decemlineata)有高毒效。发酵性能良好。YM03粉剂田间防治马铃薯甲虫有高效。稀释400倍喷雾,防治效果达94.6%。     

苏云金杆菌伴孢晶体的研究进展

苏云金杆菌能产生伴孢晶体蛋白,又叫δ—内毒素,对鳞翅目、双翅目、甚至鞘翅目的许多种昆虫幼虫有毒性。这类蛋白在芽孢形成期大量合成。对鳞翅目昆虫有毒的伴孢晶体一般为双金字塔形,蛋白质分子量为1.3×10~5道尔顿,对双翅目昆虫有毒的伴孢晶体一般为不规则的立方体,蛋白质分子量6.5×10~4道尔顿。对鞘翅目昆虫有毒的伴孢晶体一般为扁平长方形,蛋白质分子量为6.4×10~4道尔顿。对毒素蛋白的分离纯化办法、物理化学性质、毒理以及其它方面的研究现状做了介绍。     

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。     

球形芽孢杆菌C3-41二元毒素基因在苏云金芽孢杆菌以色列亚种中的克隆和表达

球形芽孢杆菌C3-41是我国分离的一株对蚊幼虫有毒杀作用的高毒力菌株,对库蚊、按蚊幼虫的毒性高于2362菌株,Southern杂交证明C\-3\|41总DNA中35Kb HindIII片段上带有419和514kD二元毒素基因,该片段由3479个核苷酸组成,核苷酸序列同2362菌株的二元毒素基因序列完全相同。含二元毒素基因的重组质粒pCW\|1和pCW\|2能在大肠杆菌中表达产生二元毒蛋白,但表达量低,重组子杀蚊毒性低。无晶体型苏云金芽孢杆菌以色列亚种重组子在其芽孢形成中能产生以晶体形式存在的二元毒素蛋白,其全发酵液和纯化晶体蛋白的杀蚊活性与C\-3\|41相近。     

苏云金芽孢杆菌的一个新亚种(Btsubsp.sinensis)

198 9年自云南昆明市石林的红棕壤中分离到数株苏云金芽孢杆菌 (Ｂａｃｉｌｌｕｓｔｈｕｒｉｎｇｉｅｎ ｓｉｓ,Ｂｔ)菌株[1] ,对其中的一株ＹＫ30 0 4进行了生物学特性、杀虫特性研究及分类鉴定。1　材料与方法1.1　供鉴定的Ｂｔ菌株由云南昆明市石林的红棕壤中分离的苏云金芽孢杆菌ＹＫ30 0 4菌株。1.2　标准Ｂｔ菌株血清型Ｈ1 Ｈ4 1、Ｈ4 4 Ｈ55及Ｈ57 Ｈ69标准Ｂｔ菌株由法国巴斯德研究院ＤｒＬｅｃａｄｅｔ提供 ,其余为本实验室保存。1.3　生物测定用昆虫小菜蛾 (Ｐｌｕｔｅｌｌａｘｙｌｏｓｔｅｌｌａ) 3龄幼虫 ;斜纹夜盗蛾 (Ｐｒ…     

Comparative toxicity of Bacillus thuringiensis var. israelensis crystal proteins in vivo and in vitro

Bacillus thuringiensis var. israelensis crystal proteins were purified by FPLC on a Mono Q column to yield 130, 65, 28, 53, 30-35 and 25 kDa proteins. All the purified proteins killed Aedes aegypti larvae after citrate precipitation, but the 65 kDa protein was the most toxic. A precipitated mixture of 27 and 130 kDa proteins was almost as toxic as solubilized crystals. In assays against a range of insect cell lines, the activated form (25 kDa) of the 27 kDa protein was generally cytotoxic with the lowest LC50 values in vitro. By contrast, the activated forms of the 130 kDa and 65 kDa protoxins (53 kDa and 30-35 kDa proteins, respectively) were much more specific than the 25 kDa protein in their action on dipteran cells, and each showed a unique toxicity profile which, in the case of the 130 kDa preparation, was restricted to Anopheles and Culex cell lines.     

Bacillus thuringiensis subspecies huazhongensis, serotype H40, isolated from soils in the People's Republic of China

DAI JINGYUAN, YU LING, WANG BO, LUO XIXIA, YU ZINIU AND M.-M. LECADET. 1996. Two isolates (YBt-981 and Ybt-978) of Bacillus thuringiensis were isolated from soil samples from Shanxi and Neimeng provinces in China. The isolates produced small and irregular parasporal inclusions that were not toxic to larvae of Culex fatigans, Culex pipiens, Anopheles sinensis, Aedes aegypti, Spodoptera littoralis, Spodoptera exigua, Plutella xylostella and Bombyx mori . H-antigens from isolates of YBt-981 and YBt-978 differed from those of the known B. thuringiensis H1 to H39 serotypes. YBt-981 and YBt-978 had the same H-antigens and the same biochemical characters. The two isolates were identified as a new serotype designated H40. The name of B. thuringiensis serovar huazhongensis for this new subspecies represented by YBt-981 and YBt-978 is proposed.     

Cloning and characterization of a novel gene cry9Ec1 encoding lepidopteran-specific parasporal inclusion protein from a Bacillus thuringiensis serovar galleriae strain

A novel delta-endotoxin gene from a lepidopteran-specific Bacillus thuringiensis serovar galleriae strain was cloned, and the full sequence of the cry gene was determined. The cloned 6.5-kb DNA fragment included the full sequence of the cry gene and three open reading frames located upstream of the cry gene. The gene, designated cry9Ec1, encodes a polypeptide of 1154 amino acid residues with a predicted molecular weight of 130 237. The deduced amino acid sequence of the Cry9Ec1 protein had the highest homology (77.7%) with the Cry9Ea1 protein when compared with existing Cry proteins. The expression, in an acrystalliferous B. thuringiensis strain, of the cry9Ec1 gene was high when controlled by the cyt1A2 promoter, leading to the formation of large spherical inclusions. The purified crystals from the recombinant strain were toxic when tested against two lepidopteran species, Bombyx mori and Plutella xylostella. However, the Cry9Ec1 protein gave no toxicity against Spodoptera litura, Spodoptera exigua, Plodia interpunctella, Helicoverpa zea, and Culex pipiens molestus.     

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.     

Bacillus thuringiensis serovar higo (flagellar serotype 44), a new serogroup with a larvicidal activity preferential for the anopheline mosquito

M. OHBA, H. SAITOH, K. MIYAMOTO, K. HIGUCHI AND E. MIZUKI. 1995. Eight strains of Bacillus thuringiensis , isolated in Japan, formed spherical parasporal inclusions and exhibited low to moderate larvicidal activities for two mosquito species, Anopheles stephensi and Culex pipiens molestus , but not for another dipteran, Telmatoscopus albipunctatus , or two lepidopterans, Bombyx mori and Hyphantria cunea . The anopheline toxicity (LC₅₀= 6.3 μg ml^-1) was > 10 times greater than the activity on the Culex mosquito. These strains were assigned to a previously undescribed flagellar (H) antigenic group. On the basis of the representative strain, 92–KU-137–4, a serogroup with H antigen 44, Bacillus thuringiensis serovar higo was established as new.     

Isolation of a non-insecticidal Bacillus thuringiensis strain belonging to serotype H8a8b

Bacillus thuringiensis strains non-toxic to Lepidoptera, Bombyx mori and Diptera, Culex pipiens pallens larvae were isolated from Korean soil samples during an investigation of B. thuringiensis isolates highly toxic to insect pests. One of these isolates, NTB-88, produces parasporal inclusions about 138 kDa in size and is non-toxic to 19 insect species of three orders, Lepidoptera, Diptera and Coleoptera, even though it is highly susceptible to tryptic cleavage. Study of flagellar (H) antibodies of 33 B. thuringiensis strains revealed that NTB-88 has an H antigen identical with that of subsp. morrisoni (serotype 8a8b). Comparison of parasporal inclusion proteins and plasmid DNA patterns of strain NTB-88 with B. thuringiensis subsp. morrisoni HD-12 and B. thuringiensis subsp. morrisoni PG-14 showed that the isolate is a novel non-insecticidal B. thuringiensis strain belonging to serotype 8a8b.     

Dissolution and Degradation of Bacillus thuringiensis delta-Endotoxin by Gut Juice Protease of the Silkworm Bombyx mori

The dissolution and degradation of dagger-endotoxin (crystal) of Bacillus thuringiensis subsp. kurstaki strain HD-1 were investigated. Crystals were dissolved in 0.1 M phosphate-carbonate-NaOH buffer at pH > 12. Swelling of crystals occurred in the buffer between pH 10 and 11, and crystals dissolved in the same buffer supplemented with gut juice protease of the silkworm Bombyx mori. The proteolytic dissolution of crystals occurred after a time lag of several minutes in 0.1 M carbonate-NaOH buffer, pH 10.2. The time lag was not observed when crystals were suspended in the buffer for 30 min before the addition of protease. After the dissolution of the crystals and further degradation of the solubilized protein, the appearance of a toxic protein with a molecular weight of 59,000, designated P-59, was observed. Lower-molecular-weight peptides (less than 40,000) showed no toxicity to the silkworm larvae on feeding. Digestion of the 120,000-dalton subunit of the crystal by gut juice protease also produced P-59. These observations suggest the occurrence of a similar process in vivo, i.e., the swelling of crystals due to the alkalinity of gut juice and the production of P-59, dependent on the hydrolysis of swollen crystals by gut juice protease.