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。     

苏云金杆菌伴孢晶体的蛋白质和抗蛋白酶多肽及其毒力特性

以SDS-聚丙烯酰胺凝胶电泳分析了19种苏云金杆菌伴孢晶体的蛋白质和抗胰蛋白酶多肽。以鳞翅目的家蚕和双翅目的致倦库蚊进行了毒力测定。根据蛋白质和抗酶多肽的特性,19种晶体可分为7个类型。晶体蛋白质的组成、溶解特性及抗酶多肽的性质与其对两种昆虫的毒力特性密切相关。含分子量为130—138 kD(千道尔顿)或130—138 kD及60一65kD蛋白质,抗蛋白酶多肽(PRP)为68—75kD的晶体,对家蚕高毒,但绝大部分对库蚊无毒或微毒。含3种以上蛋白质(15一138kD),抗酶多肽为35—65kD的晶体,对库蚊有强烈毒性,对家蚕则无毒。缺少135kD蛋白质的两种晶体对家蚕低毒。HD一282和L—14晶体的P1蛋白质中同时含有杀鳞翅目和杀蚊毒素。讨论了伴孢晶体的蛋白质结构及结构与毒力之间的关系、晶体蛋白质及毒性肽的性质在菌株定向筛选和遗传工程研究中的意义。     

棉铃虫和甜菜夜蛾中肠丝氨酸蛋白酶活性测定以及活化、降解Cry1Ac毒素分析

昆虫中肠对Bt原毒素活化与对活化毒素降解的变化被认为是害虫对Bt产生的机制之一,研究比较棉铃虫Helicoverpa armigera(Hübner)与甜菜夜蛾Spodoptera exigua(Hübner)的中肠液、BBMV蛋白酶的活性,通过SDS-PAGE分析2种昆虫对原毒素的活化速度与对活化毒素的降解速度。2种昆虫的中肠液蛋白酶活性均显著高于BBMV蛋白酶活性,中肠液与BBMV均能迅速活化原毒素并继续降解活化后的毒素,与中肠液相比,BBMV对原毒素的活化与对活化毒素的降解均慢于中肠液,甜菜夜蛾对毒素的活化与降解又慢于棉铃虫。另外,还测定抑制剂对中肠液蛋白酶活性的抑制作用,结果表明,各抑制剂对棉铃虫和甜菜夜蛾相应酶活性的抑制表现出相同的趋势,TLCK对丝氨酶蛋白酶具较好的抑制作用,而PMSF对胰蛋白酶的抑制作用次之,TPCK对胰凝乳蛋白酶的抑制作用较弱。     

棉铃虫和甜菜夜蛾中肠丝氨酸蛋白酶活性测定以及活化、降解CrylAc毒素分析

昆虫中肠对Bt原毒素活化与对活化毒素降解的变化被认为是害虫对Bt产生的机制之一,研究比较棉铃虫Helicoverpa armigern（Hǔbner）与甜菜夜蛾Spodoptera exigm（Hǔbner）的中肠液、BBMV蛋白酶的活性,通过SDS-PAGE分析2种昆虫对原毒素的活化速度与对活化毒素的降解速度。2种昆虫的中肠液蛋白酶活性均显著高于BBMV蛋白酶活性,中肠液与BBMV均能迅速活化原毒素并继续降解活化后的毒素,与中肠液相比,BBMV对原毒素的活化与对活化毒素的降解均慢于中肠液,甜菜夜蛾对毒素的活化与降解又慢于棉铃虫。另外,还测定抑制剂对中肠液蛋白酶活性的抑制作用,结果表明,各抑制剂对棉铃虫和甜菜夜蛾相应酶活性的抑制表现出相同的趋势,TLCK对丝氨酶蛋白酶具较好的抑制作用,而PMSF对胰蛋白酶的抑制作用次之,TPCK对胰凝乳蛋白酶的抑制作用较弱。     

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。     

复方苦参植物素水剂对十字花科蔬菜几种害虫的活性及田间防效

室内毒力测定结果表明,复方苦参植物素对菜蚜(若蚜)、菜青虫有很高的毒力,LC50分别为1．02mg／L和1．37mg／L,对斜纹夜蛾幼虫的毒力较低,LC50为11．65mg／L。复方苦参植物素不杀斜纹夜蛾的卵,不影响卵的孵化率,但对初孵幼虫有较好的杀伤效果。斜纹夜蛾幼虫随着虫龄的增大,对复方苦参植物素的敏感性下降。复方苦参植物素100mg／L,斜纹夜蛾幼虫非选择性和选择性拒食作用分别为96．86％和78．97％,作用方式以触杀作用为主,胃毒作用为辅。田间试验表明,复方苦参植物素200～300倍,对菜青虫和菜蚜的防效在90％左右,持效期10d。     

昆虫中肠液性质对苏云金芽孢杆菌伴孢晶体毒力的影响

综述了昆虫中肠液性质对苏云金芽孢杆菌Bacillus thuringiensis伴孢晶体毒力的影响。中肠液的酸碱度和蛋白酶是影响伴孢晶体溶解与原毒素活化的两大因素。中肠液的酸碱度不仅影响到伴孢晶体的溶解速度,还影响到各种蛋白酶的活性表现;而蛋白酶则直接参与了原毒素的活化,其组成与活性影响着原毒素的活化速度和杀虫专一性。因中肠液蛋白水解能力过高而导致原毒素的过度降解是某些昆虫对苏云金芽孢杆菌低度敏感的主要原因,而中肠液对原毒素活化能力的降低则与昆虫抗性的形成有关。此外,中肠液的沉淀作用及其它生理生化特性也影响着原毒素毒力的正常发挥。     

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。     

棉铃虫中肠类胰蛋白酶的部分纯化和性质测定及其对苏云金杆菌δ—内毒素的降解作用

将棉铃虫Helicoverpa armingera中肠液经SephadexG-75、DEAE-SephadexA-50阴离子交换和CM-SephadexC-50阳离子交换柱层析进行分离纯化,得到部分纯化的24.5kD蛋白,该蛋白可以水解胰蛋白酶的专性底物BApNA和TAME,不能水解胰凝乳蛋白酶的专性底物BTEE,蛋白酶抑制剂的抑制试验显示,TLCK、PMSF、STI及SBBI均可显著抑制该蛋白对BApNA的水解作用,而TPCK无抑制作用,由此推断,24.5kD蛋白是棉铃虫的类胰蛋白酶。以BApNA为底物,类胰蛋白酶的最适pH为10.5-11.0。SephadexG-75分离得到了4个洗脱峰,电泳结果显示,各洗脱峰均能使苏云金杆菌库斯塔克变种Bacillus thuringiensis var.kurstakiHD-1原毒素降解为60.5kD的活力片段,峰Ⅳ虽然不能水解BApNA和TAME,但仍可对原毒素进行降解,而类胰蛋白酶活力最高的峰Ⅱ已将60.5kD蛋白完全降解,当减少用量至1/3时,才出现60.5kD蛋白。因此,苏云金杆菌δ-内毒素在棉铃虫中肠内的降解有多种蛋白酶参与,类胰蛋白酶在其中起重要作用,CM-SephadexC-50进一步分离得到的类胰蛋白酶可对原毒进行降解,其降解作用受到TLCK、PMSF、STI及SBBI的显著抑制,不受TPCK的影响。     

Insecticidal properties of a crystal protein gene product isolated from Bacillus thuringiensis subsp. kenyae.

A protoxin gene, localized to a high-molecular-weight plasmid from Bacillus thuringiensis subsp. kenyae, was cloned on a 19-kb BamHI DNA fragment into Escherichia coli. Characterization of the gene revealed it to be a member of the CryIE toxin subclass which has been reported to be as toxic as the CryIC subclass to larvae from Spodoptera exigua in assays with crude E. coli extracts. To directly test the purified recombinant gene product, the gene was subcloned as a 4.8-kb fragment into an expression vector resulting in the overexpression of a 134-kDa protein in the form of phase-bright inclusions in E. coli. Treatment of solubilized inclusion bodies with either trypsin or gut juice from the silkworm Bombyx mori resulted in the appearance of a protease-resistant 65-kDa protein. In force-feeding bioassays, the purified activated protein was highly toxic to larvae of B. mori but not to larvae of Choristoneura fumiferana. In diet bioassays with larvae from S. exigua, the purified protoxin was nontoxic. However, prior activation of the protoxin by tryptic digestion resulted in the appearance of some toxic activity. These results demonstrate that this new subclass of protein toxin may not be useful for the control of Spodoptera species as previously reported. Hierarchical clustering of the nine known lepidopteran-specific CryI toxin subclasses through multiple sequence alignment suggests that the toxins fall into four possible subgroups or clusters.     

Specificity of Bacillus thuringiensis var. colmeri insecticidal delta-endotoxin is determined by differential proteolytic processing of the protoxin by larval gut proteases

The native crystal delta-endotoxin produced by Bacillus thuringiensis var. colmeri, serotype 21, is toxic to both lepidopteran (Pieris brassicae) and dipteran (Aedes aegypti) larvae. Solubilization of the crystal delta-endotoxin in alkaline reducing conditions and activation with trypsin and gut extracts from susceptible insects yielded a preparation whose toxicity could be assayed in vitro against a range of insect cell lines. After activation with Aedes aegypti gut extract the preparation was toxic to all of the mosquito cell lines but only one lepidopteran line (Spodoptera frugiperda), whereas an activated preparation produced by treatment with P. brassicae gut enzymes or trypsin was toxic only to lepidopteran cell lines. These in vitro results were paralleled by the results of in vivo bioassays. Gel electrophoretic analysis of the products of these different activation regimes suggested that a 130-kDa protoxin in the native crystal is converted to a 55-kDa lepidopteran-specific toxin by trypsin or P. brassicae enzymes and to a 52-kDa dipteran toxin by A. aegypti enzymes. Two-step activation of the 130-kDa protoxin by successive treatment with trypsin and A. aegypti enzymes further suggested that the 52-kDa dipteran toxin is derived from the 55-kDa lepidopteran toxin by enzymes specific to the mosquito gut. Confirmation of this suggestion was obtained by peptide mapping of these two polypeptides. The native crystal 130 kDa delta-endotoxin and the two insect-specific toxins all cross-reacted with antiserum to B. thuringiensis var. kurstaki P1 lepidopteran toxin. Preincubation of the two activated colmeri toxins with P1 antiserum neutralized their cytotoxicity to both lepidopteran and dipteran cell lines.     

Processing of delta-endotoxin from Bacillus thuringiensis subsp. kurstaki HD-1 and HD-73 by gut juices of various insect larvae.

Midgut juices were prepared from Adoxophyes sp., smaller tea tortrix (STT); Bombyx mori, silkworm (SW); Spodoptera litura, common cutworm (CCW); Plutella xylostella, diamondback moth (DBM); and Musca domestica, housefly (HF) and immobilized onto Sepharose 4B. delta-Endotoxins (ICPs) from Bacillus thuringiensis subsp. kurstaki HD-1 and HD-73 were digested by these immobilized gut juice proteases. All gut juices tested derived relatively proteolytic resistant cores from ICP. The molecular sizes of these cores, about 55 kDa in SDS-PAGE, were resulted. In the case of CCW, however, digestion was very strong and only 1/20 concentration of core protein remained relative to other digests. The N-terminal amino acid sequencing of the core proteins showed that they were truncated at the very end of the N-terminus of protoxin, CryIA, at different sites. Although housefly larvae were completely insensitive to active toxin, the gut juice produced the core, suggesting that the housefly may lack the binding sites for the core-active toxin.     

Comparative study of Bacillus thuringiensis Cry1Ia and Cry1Aa delta-endotoxins: Activation process and toxicity against Prays oleae

Cry1Ia and Cry1Aa proteins exhibited toxicities against Prays oleae with LC₅₀ of 189 and 116 ng/cm², respectively. The ability to process Cry1Ia11 protoxin by trypsin, chymotrypsin and P. oleae larvae proteases was studied and compared to that of Cry1Aa11. After solubilization under high alkaline condition (50 mM NaOH), Cry1Aa11 was converted into a major fragment of 65 kDa, whereas Cry1Ia11 protoxin was completely degraded by P. oleae larvae proteases and trypsin and converted into a major fragment of 70 kDa by chymotrypsin. Using less proteases of P. oleae juice, the degradation of Cry1Ia11 was attenuated. When the solubilization (in 50 mM Na₂CO₃ pH 10.5 buffer) and activation were combined, Cry1Ia11 was converted into a proteolytic product of 70 kDa after 3 h of incubation with trypsin, chymotrypsin and P. oleae juice. These results suggest that the in vivo solubilization of Cry1Ia11 was assured by larval proteases after a swelling of the corresponding inclusion due to the alkalinity of the larval midgut.     

Bacillus thuringiensis crystal delta-endotoxin: role of proteases in the conversion of protoxin to toxin

The conversion of delta-endoprotoxins of Bacillus thuringiensis to active toxins is mediated by trypsin, insect gut (exogenous) and bacterial (endogenous) proteases. The biochemical aspects of exogenous and endogenous proteases involved in the conversion of protoxin to toxin are reviewed. Perhaps, these proteases also play a role in influencing the host range of toxin and in the development of resistance to toxin.     

Analysis of the molecular basis of insecticidal specificity of Bacillus thuringiensis crystal delta-endotoxin.

The mechanism of action and receptor binding of a dual-specificity Bacillus thuringiensis var. aizawai ICl delta-endotoxin was studied using insect cell culture. The native protoxin was labelled with 125I, proteolytically activated and the affinity of the resulting preparations for insect cell-membrane proteins was studied by blotting. The active preparations obtained by various treatments had characteristic specificity associated with unique polypeptides, and showed affinity for different membrane proteins. The lepidopteran-specific preparation (trypsin-treated protoxin containing 58 and 55 kDa polypeptides) bound to two membrane proteins in the lepidopteran cells but none in the dipteran cells. The dipteran-specific preparation (protoxin treated sequentially with trypsin and Aedes aegypti gut proteases, containing a 53 kDa polypeptide) bound to a 90 kDa membrane protein in the dipteran (A. aegypti) cells but bound to none in the lepidopteran cells or Drosophila melanogaster cells. The toxicity of trypsin-activated delta-endotoxin was completely inhibited by preincubation with D-glucose, suggesting a role for this carbohydrate in toxin-receptor interaction. The toxicity was also decreased by osmotic protectants to an extent proportional to their viscometric radius. These results support a proposal that initial interaction of toxin with a unique receptor determines the specificity of the toxin, following which cell death occurs by a mechanism of colloid osmotic lysis.     

Processing of Cry1Ab delta-endotoxin from Bacillus thuringiensis by Manduca sexta and Spodoptera frugiperda midgut proteases: role in protoxin activation and toxin inactivation

Activation of Cry protoxins is carried out by midgut proteases. This process is important for toxicity and in some cases for specificity. Commercial proteases have been used for in vitro protoxin activation. In the case of Cry1A protoxins, trypsin digestion generates a toxic fragment of 60–65 kDa. Here, we have analyzed the in vitro and in vivo activation of Cry1Ab. We found differences in the processing of Cry1Ab protoxin by Manduca sexta and Spodoptera frugiperda midgut proteases as compared to trypsin. Midgut juice proteases produced two additional nicks at the N-terminal end removing helices 1 and 2a to produce a 58 kDa protein. A further cleavage within domain II splits the toxin into two fragments of 30 kDa. The resulting fragments were not separated, but instead coeluted with the 58 kDa monomer, in size-exclusion chromatography. To examine if this processing was involved in the activation or degradation of Cry1Ab toxin, binding, pore formation, and toxicity assays were performed. Pore formation assays showed that midgut juice treatment produced a more active toxin than trypsin treatment. In addition, it was determined that the 1 helix is dispensable for Cry1Ab activity. In contrast, the appearance of the 30 kDa fragments correlates with a decrease in pore formation and insecticidal activities. Our results suggest that the cleavage in domain II may be involved in toxin inactivation, and that the 30 kDa fragments are stable intermediates in the degradation pathway.     

Processing of Cry1Ab δ-endotoxin from Bacillus thuringiensis by Manduca sexta and Spodoptera frugiperda midgut proteases: role in protoxin activation and toxin inactivation

Activation of Cry protoxins is carried out by midgut proteases. This process is important for toxicity and in some cases for specificity. Commercial proteases have been used for in vitro protoxin activation. In the case of Cry1A protoxins, trypsin digestion generates a toxic fragment of 60–65 kDa. Here, we have analyzed the in vitro and in vivo activation of Cry1Ab. We found differences in the processing of Cry1Ab protoxin by Manduca sexta and Spodoptera frugiperda midgut proteases as compared to trypsin. Midgut juice proteases produced two additional nicks at the N-terminal end removing helices α1 and α2a to produce a 58 kDa protein. A further cleavage within domain II splits the toxin into two fragments of 30 kDa. The resulting fragments were not separated, but instead coeluted with the 58 kDa monomer, in size-exclusion chromatography. To examine if this processing was involved in the activation or degradation of Cry1Ab toxin, binding, pore formation, and toxicity assays were performed. Pore formation assays showed that midgut juice treatment produced a more active toxin than trypsin treatment. In addition, it was determined that the α1 helix is dispensable for Cry1Ab activity. In contrast, the appearance of the 30 kDa fragments correlates with a decrease in pore formation and insecticidal activities. Our results suggest that the cleavage in domain II may be involved in toxin inactivation, and that the 30 kDa fragments are stable intermediates in the degradation pathway.     

Proteolytic processing of Bacillus thuringiensis toxin Cry1Ab in rice brown planthopper, Nilaparvata lugens (Stål)

To understand the low toxicity of Cry toxins in planthoppers, proteolytic activation of Cry1Ab in Nilaparvata lugens was studied. The proteolytic processing of Cry1Ab protoxin by N. lugens midgut proteases was similar to that by trypsin activated Cry1Ab. The Cry1Ab processed with N. lugens midgut proteases was highly insecticidal against Plutella xylostella. However, Cry1Ab activated either by trypsin or the gut proteases of the brown planthopper showed low toxicity in N. lugens. Binding analysis showed that activated Cry1Ab bound to brush border membrane vesicles (BBMV) from N. lugens at a significantly lower level than to BBMV from P. xylostella.