PH对苏去金杆菌晶体致病性的影响

报道了以苏去金杆菌５个菌株的晶体,经粘虫和黄粉虫幼虫的肠液在ＰＨ为６．４、７．４、８．４和９．４的缓冲液中（２８℃）消化６小时后,其上清液（消化部分）和沉淀（未消化部分）对三龄未粘虫和黄粉虫幼虫的毒力。结果表明,４５７株、ＨＤ－１株和１－４－１３株的上清液和沉淀对粘虫均有不同程度的毒效,Ｔ８４－１株和Ｔ２株的上清液和沉淀对粘虫均无毒效;而对黄粉虫幼虫,以ＰＨ９．４时的上清液和沉淀均无毒效,在ＰＨ６     

乙醇酸氧化酶纯化方法的改进

乙醇酸氧化酶（GO）是光呼吸的关键酶。但目前报道的GO的Mr和等电点等基本参数相差很大,重复性也不好[2,3,5～11]。本实验拟改进其纯化方法以期提高其重复性。材料与方法菠菜（Spinaciaoleracea）和菜心（Brassicacampestris）均购自市场。GO的提纯基本按文献2、3、6,只作添加FMN一项改进。具体操作如下：绿叶用100mmol·L－－‘的磷酸缓冲液（pH8．0）提取粗蛋白,过滤,4000Xg离心取上清液,加10％HAC调到PH53;然后以4000Xg离心取上清液,经15％～30％（NH;）iS04分步沉淀;高GO活性的沉淀蛋白用5mmol·L‘Tris－HCI（pH…     

棉铃虫为害水稻

近年来笔者从事水稻纹枯病研究工作,每3～5天即在稻田调查。1996年8月中下旬发现有虫暴露在水稻植株上为害,经鉴定确认为棉铃虫He伽ovefpaa七Wigero－－xxx－xxuo。此类报道未见,故简报如下。（l）水稻品种为粳稻（武有粳3号）;（2）调查稻田距棉田200～500m;（3）稻田中棉铃虫幼虫l～6龄均有;M幼虫均在稻株顶叶或穗部,水稻未抽穗时,幼虫啮食嫩叶叶肉,少数食成缺刻,抽穗后,幼虫多数爬至穗部咬食灌浆、乳熟稳粒;（5）千株有虫0～5头不等。1996年本市四代棉铃虫在棉田落卵量为1556粒／百株,属特大发生。笔者分析认为稻田中棉铃…     

小黑瓢虫对高氏瘤粉虱捕食作用的研究

在高氏瘤粉虱不同虫态共存的条件下,小黑标虫对高氏瘤粉虱各虫态的选择次序为卵＞1龄若虫＞2龄若虫＞3龄若虫＞4龄若虫和拟蛹,对卵的捕食率均最高,有明显的嗜好选择;小黑瓢虫幼虫捕食粉虱卵的数量,随着龄期的增长而递增,其中4龄幼虫的捕食量最大,4龄期捕食量平均为1565．42粒,占全幼虫期总食卵量的45．42％,整个幼虫期可捕食高氏瘤粉虱的卵数平均为3446．5粒。小黑瓢虫3龄幼虫对粉虱卵的捕食作用率在所给的猎物密度（1500粒／皿）条件下,随着自身密度的增加而降低。     

储藏物害虫嗜虫书虱的生物学研究(英文)

以全麦粉和酵母粉（10：1）为饲料,分别在8种温度（17．5－35℃）及5种相对湿度（50％－90％RH）条件下系统地研究了嗜虫书虱的生物学特性,获得了其在不同温、湿度组合情况下的生长发育、生存和繁殖的一系列特性和参数。结果表明,在75％RH、20－35℃范围内,世代发育历期在71．9－21．7天之间。世代发育起点温度和有效积温分别为15．7℃和460．7日·度。各虫态及世代存活率均以27．5℃时最高,温度过高或过低均不利于该虫存活。成虫平均寿命在64．4－32．4天之间。每雌平均产卵量为21．1－13．9粒。在28℃条件下,较高的相对温度有利于该虫生长发育和繁殖。相对湿度低于50％或温度低于17．5℃,该虫均不能完成世代发耷。总体来看该虫生长发育、繁殖的最适温区为28－30℃,最适相对湿度在80％—90％之间并就该虫在我国分布及为害严重的原因进行了探讨。     

用性信息素诱剂防治烟青虫的效果

本文研究了两个烟青虫性信息素诱剂配方对烟青虫Helicoverpaassulta (Guen e)成虫的诱杀效果及诱杀成虫后田间烟青虫幼虫的虫口减退率和有虫株减退率。 1型诱芯配比为Z9 1 6 :Ald与Z1 1 1 6 :Ald 1 0 0∶9 5 ;Ⅱ型诱芯配比为 1 6 :Ald和Z9 1 6 :Ald与Z1 1 1 6 :Ald( 1 9 3∶1 0 0∶70 )。结果表明 :烟青虫性信息素诱剂Ⅰ ,Ⅱ型诱芯对烟青虫成虫都有很强的诱杀效果 ,在长达 85d的时间内 ,5个Ⅰ型诱芯共诱杀了4 0 4头雄性成虫 ,5个Ⅱ型诱芯共诱杀 4 1 9头 ,二者没有明显的差异。诱蛾地烟青虫幼虫的虫口密度和有虫株率都有明显的降低 ,Ⅰ、Ⅱ型诱芯使虫口密度和有虫株率的降低幅度均在 6 6 6 7%～ 90 91 %之间。性信息素诱剂使用方便、无毒、不污染环境 ,对烟叶不产生药害 ,防治效果良好 ,具有良好的应用前景。     

康氏木霉B—7和黑曲霉X—15原生质体的形成和再生

研究了康氏木霉B－7和黑曲霉X－152株纤维素酶高产菌株的原生质体制备与再生。结果表明,采用纤维素酶、蜗牛酶、溶菌酶的混合酶液,可成功地制备2株真菌的原生质体。其中,B－7以这3种酶的配比6：5：2为最佳,X－15以8：4：2为最佳。原生质体形成的缓冲液系统均以0．2mol/L,pH6．0磷酸盐缓冲液为宜,渗透压稳定剂则分别以0．6mol/LNaCl和0．6mol/L蔗糖为宜。以菌龄18h（B-7）和16h（X－15）2株真菌的菌丝体,在37℃下酶解90min可获得最适量的原生质体,产量分别达9×106个／ml和1．9×107个／ml,且其再生率也较高,均达95％左右。     

一株高产木聚糖酶真菌的筛选及酶学性质分析

从近百份土样中,首先筛选得到具有明显半纤维素水解活性透明圈的菌株564株,其中202株的木聚糖酶活力用96孔板筛选方法进行了验证。结果显示：基于96孔深孔板测定的酶活力与初筛测得的透明圈活力趋势一致。最终筛选得到一株高产木聚糖酶的菌株ECU2023,经核糖体rDNA内部转录间隔区的序列对比,鉴定为泡盛曲霉（Aspergillus awamori）。研究获得了其最佳培养条件：pH6．0,培养时间4d,C源为30～40g/L玉米芯粉。经过（NH4）2SO4沉淀和Superdex G-75凝胶过滤层析后,其中主要木聚糖酶成分的最适反应pH为6．0,最适反应温度为50℃。     

昆虫肠道蛋白酶对苏芸金杆菌晶体作用的比较

本文以粘虫Leucania separata Walker,棉铃虫Heliothis armigera Hūbner大蜡螟Gatleria mellonella Linnaeus和黄地老虎Agrotis segeium Schiffermǖller 4种幼虫的肠道蛋白酶作用于苏芸金杆菌7404肯尼亚变种(B.t.var.kenyae)、HD-1戈尔斯德变种(B.t.var.kurstaki)7216天门变种(B.t.var.tianmensis)、75023(变种未定)、007苏芸金变种(B.t.var.thurtngiensis)、006玉米螟变种(B.t.var.ostriniae)、7417松蠋变种(B.t.var.dendrolimus)及1897以色列变种(B.t.var. israelensis)的晶体,比较了其消化产物的聚丙烯酰胺凝胶电泳图谱。对粘虫肠道蛋白酶消化的晶体形态进行了电镜观察,并对晶体消化过程中光密度的变化作了测定。非病原菌的晶体亦能被肠液中的蛋白酶消化。     

粉被虫草N~6-(2-羟乙基)腺苷高产菌株的原生质体诱变育种

在粉被虫草（CordycepspruinosaPetch）无性型─-粉被马利娅霉（MariannaeaPruinosaLiang）原生质体形成和再生的前期研究基础上,报道了通过原生质体诱变培育高产N6－（2－羟乙基）腺苷菌株的研究结果。粉被马利娅霉Cp－14单孢子株经15W紫外灯照射后,获得一高产突变株MpM－135。经多代移植后,此突变株比出发菌株Cp－14的N6－（2－羟乙基）腺苷含量提高了14．8％。实验还表明,粉被虫草无性型菌丝提取物对枯草杆菌（Bacillussubtilis）抗紫外辐射效果与菌丝所含N6－（2－羟乙基）腺苷含量有正相关性。     

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.     

Two types of entomocidal toxins in the parasporal crystals of Bacillus thuringiensis kurstaki

Two types of entomocidal proteins of Bacillus thuringiensis kurstaki were isolated from the parasporal bodies (crystals), and their structures were compared with each other in relation to the toxic activity. When the crystals were dissociated in 2% 2-mercaptoethanol at pH 10, a protein of Mr = 135,000, called delta-endotoxin, was liberated. The crystals of a strain of B. thuringiensis kurstaki, the HD-1 strain, also released another protein in small quantities. This minor component of HD-1, which had been discovered and named mosquito factor by Yamamoto and McLaughlin (T. Yamamoto and R. E. McLaughlin (1981) Biochem. Biophys. Res. Commun. 103, 414-421) because of its toxicity to mosquito larvae, could be liberated selectively from the crystals by alkali treatment without any thiol reagent at pH 11. Electron microscopic observation suggested that the bipyramidal crystal is composed of a homogeneous component, presumably the delta-endotoxin, and the mosquito factor is not within the crystal matrix. The liberated toxins, including the mosquito factor, were purified by Sephacryl S-300 column chromatography and activated by proteinases obtained from gut juice of the cabbage looper (Trichoplusia ni). The activated toxins were characterized by peptide mapping using techniques of HPLC and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Peptide mapping revealed that the mosquito factor is a protein distinctly different from the delta-endotoxin. Furthermore, a comparison between two strains of B. thuringiensis kurstaki indicated that minor differences in the structure of the delta-endotoxins, in particular the differences in their proteinase-resistant region, caused significant variations in their toxicity to susceptible insects.     

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

The dissolution and degradation of †-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.     

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。     

EFFECT OF DISSOLUTION AND DEGRADATION OF BAClLUlS THURlNGIENSlS PARASPORAL CRYSTALS ON TOXICITY TO COTTON BOLLWORM*

Abstract The effect of dissolution and buffer pH values on the proteolysis of Bacillus thuringiensis subvar. kurstaki HD-1 parasporal crystals were studied by SDS-PAGE. Dissolution made crystal much easier to be digested by larval Helicoverpa armigera midgut proteases; the rate of proteolysis of protoxin was faster than that of the crystal. Incubated with larval midgut juice, bovine trypsin and chymotrypsin for 17 h at 30°C, the proteolytic process of crystals deepened as the pH of buffer rose from 6. 9 to 10.5; the complete degradation of crystals occurred at pH 10.5, and the active domains were similar, with molecular weight of 60.5±1.26Kd, 61.6±1.16Kd, and 61.7±2.0Kd respectively. Bioassay results suggested that incubation in alkaline buffer could improve the toxicity of crystals to H. armigera, proteolysis gave no further modification to the toxicity and the toxicity was not significantly different among the products from proteolysis by larval midgut juice, bovine trypsin and chymotrypsin, Whether diets with different pH would affect the toxicity of crystals to H. armiger were also tested. Diets made by 0. 01mol.L^-1 Tris-HC1 buffer, pH 8.0 and 0.01mol*L^-1 glycine-NaOH buffer, pH 10.0, all significantly increased the toxicity of crystals and no difference between them was observed.     

Midgut proteases from Mamestra configurata (Lepidoptera: Noctuidae) larvae: characterization,cDNA cloning,and expressed sequence tag analysis

The activities of digestive protease within the midgut of Mamestra configurata (bertha armyworm) larvae were examined using specific substrates and protease inhibitors. The bulk of the activity was associated with serine proteases comprising trypsin-, chymotrypsin-, and elastase-like enzymes. At least 10-15 serine protease isozymes were detected using one-dimension gelatin gel electrophoresis. Cysteine or aspartic protease activities were not present; however, amino- and carboxypeptidase activities were associated with the midgut extract. Midgut proteases were active in the pH range of 5.0-12.0 with peaks at pH 7.5 and 11.0. In general, the middle region of the midgut exhibited a higher pH (approximately 8.0) than either the posterior or anterior regions (approximately 7.3-7.7). Moulting larvae possessed a neutral gut pH that was 0.5-1.5 units below that of feeding larvae. Degenerate PCR and expressed sequence tag (EST)-based approaches were used to isolate 30 distinct serine protease encoding cDNAs from a midgut-specific cDNA library including 8 putative trypsins, 9 chymotrypsins, 1 elastase, and 12 whose potential activities could not be determined. cDNAs encoding three amino- and two carboxypeptidases were also identified. Larvae feeding upon artificial diet containing 0.2% soybean trypsin inhibitor experienced a significant delay in development.     

Serotypes of verocytotoxigenic Escherichia coli isolated from cattle and buffalo calf diarrhoea

Abstract Parasporal crystals of the recently isolated Bacillus thuringiensis var. tenebrionis are toxic for coleopteran larvae. Unlike those of other strains they are soluble either in aqueous solutions of NaBr at neutral pH or in water after titration to pH values above pH 10.0. The dissolved crystal protein readily forms crystals after removal of the salt or neutralization. The crystal protein was not found to differ much in the amino acid composition from other crystal proteins. The parasporal crystals are composed of subunits of M _r 68 000 which are not linked by disulfide bridges.     

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.     

Single cysteine substitution in Bacillus thuringiensis Cry7Ba1 improves the crystal solubility and produces toxicity to Plutella xylostella larvae

Many Bacillus thuringiensis isolates have no demonstrated toxicity against insects. In this study, a novel holotype crystal protein gene cry7Ba1 was isolated from a 'non-insecticidal'B. thuringiensis strain YBT-978. The Cry7Ba1 protein showed high toxicity against Plutella xylostella larvae after the crystals were dissolved at pH 12.5, suggesting that the 'non-insecticidal' properties of this protein were due to insolubility in the normal insect midgut pH environment. After the C-terminal half of Cry7Ba1 was replaced by that of Cry1Ac or Cry1C proteins, the recombinant protein inclusions could be dissolved at pH 9.5, and exhibited high toxicity against P. xylostella larvae. This result proved the insolubility of Cry7Ba1 crystal was determined by the structure of its C-terminal half. Further, six mutations were constructed by substituting cysteine residues with serine. Solubility studies showed that the crystals from mutants C697S, C834S and C854S could be dissolved at lower pH (10.5, 9.5 and 11.5 respectively). Bioassays showed that crystals from mutant C834S were toxic to P. xylostella larvae. Our discoveries suggest that a single cysteine residue located in the C-terminal half of the protein determines the solubility and toxicity of some nontoxic crystal proteins. This study provides a strategy to isolate novel insecticidal crystal protein genes from 'non-insecticidal'B. thuringiensis strains.     

Host plant resistance in romaine lettuce affects feeding behavior and biology of Trichoplusia ni and Spodoptera exigua (Lepidoptera: Noctuidae)

Lettuce quality and yield can be reduced by feeding of several lepidopterous pests, particularly cabbage looper, Trichoplusia ni (Hübner), and beet armyworm, Spodoptera exigua (Hübner) (Lepidoptera: Noctuidae). Host plant resistance to these insects is an environmentally sound adjunct to conventional chemical control. In this study we compared the survival, development, and feeding behavior of cabbage looper and beet armyworm on two romaine lettuce cultivars, resistant 'Valmaine' and susceptible 'Tall Guzmaine'. Larval mortality of both species was significantly higher on resistant Valmaine than on susceptible Tall Guzmaine. The average weight per larva after feeding for 1 wk on Tall Guzmaine plants was 6 times (beet armyworm) and 2 times (cabbage looper) greater than that of larvae feeding on Valmaine plants. Significant reduction in larval growth on Valmaine compared with that on Tall Guzmaine resulted in a 5.9- (beet armyworm) and 2.6-d (cabbage looper) increase in larval duration and almost a 1-d increase in pupal duration. Average pupal and adult weights and successful pupation of cabbage looper and beet armyworm were reduced on Valmaine compared with Tall Guzmaine. The sex ratio of progeny did not deviate from 1:1 when larvae were reared on either Valmaine or Tall Guzmaine. The fecundity of cabbage looper and beet armyworm adults that developed from larvae reared on Valmaine was about one-third that of adults from Tall Guzmaine, but adult longevity did not significantly differ on the two lettuce cultivars. The two insect species showed different feeding preferences for leaves of different age groups on Valmaine and Tall Guzmaine. Cabbage loopers cut narrow trenches on the leaf before actual feeding to block the flow of latex to the intended site of feeding. In contrast, beet armyworms did not trench. The different feeding behavior of the two species on Valmaine may explain the superior performance of cabbage looper compared with beet armyworm.