一株蝗虫病原菌的分离和鉴定

从内地黄脊竹蝗自然病死虫尸内分离到一种病原菌,其纯培养物经KOCK病症律证明,并按《伯杰细菌鉴定手册》(第九版)的方法进行了生物学测定、生理生化实验,并测定了其DNA中G+Cmol含量为63.73％,确定该病原物为类产碱假单胞菌(Pseudomonas pseudoalcaligenes)。经生物测定,该菌对草地蝗虫和黄脊竹蝗有较强的感染力,对草地其他害虫也有一定的感染力。     

嗜麦芽假单胞菌酪氨酸酶基因在类产碱假单胞菌中的转移与表达

类产碱假单胞菌 (Pseudomonaspseudoalcaligenes)是从盐城市农村粪池中采集的自然病死蝇蛆体内分离的具有显著杀蛆作用的细菌 ,野外使用易受阳光中紫外线的影响而失活。黑色素具有很强的抗辐射作用。将构建的含有嗜麦芽假单胞菌酪氨酸酶基因的质粒pWSY导入类产碱假单胞菌体内 ,使后者获得了稳定产生黑色素的能力。Southern杂交实验证实酪氨酸酶基因来源于嗜麦芽假单胞菌。SDS PAGE电泳显示该重组子体内额外表达了一分子量约为 1 8kD的蛋白 ,该蛋白很可能就是重组子表达的酪氨酸酶。经测定 ,重组子抗辐射作用明显增强 ,有效杀蛆时间显著延长 ,对畜、禽安全     

类产碱假单胞菌降解聚乙烯醇阶段研究成果技术鉴定会

从工厂含聚乙烯醇废水中筛选到一株对高分子化合物聚乙烯醇(Polyvinul Alcokol)有较强降解作用的类产碱假单胞菌D 8菌株。该菌株在聚乙烯醇为唯一碳源的培养基中培养     

产碱假单胞菌一个新变种

关于假单胞菌的研究,已有许多文献报道。我们于1979—1980年从黑斑泥鳅肠腔内先后分离出假单胞菌83株。经鉴定该菌与产碱假单胞菌(Pseudomonas alcaligenes)很相似,但也有些不同。故认为该菌是产碱假单胞     

类产碱假单胞菌杀虫蛋白的激光拉曼光谱研究

类产碱假单胞菌是一种新发现的昆虫病原微生物,其代谢产生的杀虫蛋白对蝗虫具有较强的毒杀作用,经由杀虫蛋白的水和重水溶液的拉曼光谱,按照Lippert的方法计算它的二级结构含量,β折叠含量为58%,无规卷曲为34%,侧链C—C—S—S—C—C构型为反式-扭曲-反式.它的酪氨酸残基大部分暴露在分子表面,小部分埋藏在疏水环境中.并讨论了杀虫蛋白结构有可能导致的杀虫机理.     

类产碱假单胞菌谷氨酸脱氢酶的纯化和性质

谷氨酸脱氢酶（Ｇｌｕｔａｍａｔｅｄｅｈｙｄｒｏｇｅｎａｓｅ,ＧＤＨ）可逆催化谷氨酸脱氨生成α酮戊二酸和氨,是一种依赖ＮＡＤ（Ｐ）的脱氢酶。其分子形式主要为六聚体或四聚体,大多数ＧＤＨ为六聚体［１］。该酶广泛存在于原核生物和真核生物,在氮、碳代谢的联系方面发挥重要作用。至今已有很多细菌ＧＤＨ得到详细研究［２～４］,但未见类产碱假单胞菌ＧＤＨ的研究报道。本文通过研究类产碱假单胞菌ＧＤＨ的纯化和性质,为揭示该菌的氮、碳代谢机制奠定基础。１　材料和方法１－１　细菌培养液体培养基为含柠檬酸和氯化铵的微量…     

类产碱假单胞菌杀虫蛋白对蝗虫能量代谢的影响

电镜直接观察结果显示,在类产碱假单胞菌杀虫蛋白作用下,蝗虫中肠细胞线粒体病变严重,多数呈现膨胀,有嵴断裂等现象。氧电极技术分析发现病变后蝗虫胃、肠细胞线粒体氧化磷酸化作用减弱,ATP产生减少,能量代谢被抑制,但ATPase活力基本不受影响,即蝗虫体内能量需求仍可维持正常水平,由此导致蝗虫体内能量供应不足而死亡。     

Pm—1蝗虫病原菌的鉴定

从蝗虫自然病死虫尸上分离到的一株病原菌,按照《Bergey's Manual of Systematic Bacterio logy》鉴定方法,作了形态观察,生理生化和生态学实验,用Marmur和Doty的DNA热变性解链法,测得细胞中DNA的GC值含量为66.6％,对照检索表确定该菌株为产碱假单胞菌(Pseudomonas alcaligenes)。     

立体选择性脂肪酶的筛选及其在薄荷醇拆分中的应用

采用双平板透明圈方法,筛选得到1株能够选择性水解L-薄荷醇丙酸酯的微生物。通过16sDNA测序,鉴定为产碱假单胞菌Pseudomonas alcaligenes。该菌所产脂肪酶可水解4对薄荷醇丙酸酯,在35℃时绝对转化率达到50％时,表现出较高的对映体过量值（e.e.p〉99％）与非对映体过量值（d．e．p〉90％）。这一结果表明,产碱假单胞菌脂肪酶是具有工业应用前景的生物催化剂。     

类产碱假单胞菌核心杀虫蛋白基因的转化及杀虫活性

[目的]利用类产碱假单胞菌核心杀虫蛋白基因(Core Pseudomonas pseudoalcaligenes insecticidal protein gene,cppip)构建植物表达载体并转化烟草,以研究cppip在高等植物体内表达产物的活性.[方法]cppip在烟草基因组中的整合及转录通过烟草转化系T0代种子发芽的抗生素抗性分离及其T1代株系的分子检测来证明;烟草转化系后代表达产物的杀虫效果通过测定蝗虫死亡率来分析.[结果]证明了cppip能以一定拷贝数插入到烟草基因组内,并按照孟德尔遗传方式传递给后代;比较含信号肽(signal peptide sequence,SPS)和不含信号肽的类产碱假单胞菌杀虫蛋白基因(Pseudomonas pseudoalcaligenes insecticidal protein gene,ppip)表达产物的杀虫活性,发现不含SPS的ppip烟草转化系蛋白表达产物对2～3龄蝗虫幼虫的平均致死率为83.37%,并对幼虫的生长发育有明显抑制作用,而含有SPS的ppip烟草转化系蛋白表达产物对2～3龄蝗虫幼虫的平均致死率为15.65%,两者之间有着显著的差异.[结论]推测ppip的SPS会影响该基因在高等植物体内表达产物的活性,本研究结果对于高效利用ppip进行植物转化及抗虫具有重要参考价值.     

类产碱假单胞菌杀虫蛋白的N端鉴定及抗体制备

The insecticidal protein from Pseudomonas pseudoaligenes was and exotoxin which had toxicity on locusts.In order to elucidate its molecular properties an amino acid sequence,the insecticidal protein was purified from the culture supernatant by ultrafiltration,ion\|exchange chromatography and gel filtration,and showed a single band on SDS\|PAGE.Analysis of the purified insecticidal protein dentified N\|terminal sequence of ten amino acid residues.Its polyclonal antibody was also obtained by immunizing rabb…     

球形芽孢杆菌Ts—1毒蛋白的分离纯化

Bacillus sphaericus strain Ts-1 is highly insecticidal to larvae of the mosquito. It's insecticidal component is toxic proteins. The toxin was extracted from spore-crystal complexes by disruption in a Sonicator Cell Disruptor Model W-220F followed by treatment with 0.05 mol/L NaOH. Fraction recovered from chromatography of the spore-crystal complexes on column of Sephadex G-200 were assayed against mosquito larvae and the toxic fractions from gel chromatography were subjected to SDS-PAGE. The toxic proteins in B. sphaericus Ts-1 spore-crystal complex migrated in position corresponding to 42kD and 43kD. Bioassay of the two purified proteins prepared by PAGE indicated that they were all toxic to mosquito larvae. Toxic protein was further purified by DEAE-cellulose chromatography. The toxic protein with a molecular weight of 42kD was obtained.     

Cry1Ah蛋白标准物质候选物的制备与纯化

随着转基因技术的迅猛发展,转基因产品的安全性受到了广泛关注。转基因检测用有证标准物质在确保转基因产品定性、定量检测结果的可比性和可追溯性方面发挥着重要作用。但转基因蛋白质标准物质的开发相对缓慢,其中一个难点是制备高纯度的转基因蛋白质候选物。苏云金芽胞杆菌Bacillus thuringiensis cry1Ah1基因因其对亚洲玉米螟等鳞翅目害虫有很好的杀虫活性,已用于转基因抗虫作物的研制,并获得具有较好抗虫性状的转基因株系。为了研发Cry1Ah蛋白有证标准物质,亟需建立其制备及纯化体系。文中优化了利用Bt表达系统制备Cry1Ah蛋白的体系,利用离子交换色谱法和排阻色谱法逐级纯化的方法,获得了高纯度的Cry1Ah蛋白 (排阻色谱纯度：99.6%)。生物活性测定结果表明,纯化的Cry1Ah蛋白与原毒素对小菜蛾Plutella xylostella的杀虫活性没有显著差异。最后使用Edman降解法和质谱法确定了Cry1Ah蛋白活化后的氨基酸序列。综上所述,获得的Cry1Ah纯蛋白可用于蛋白质标准物质的研制。     

Cry1Ah蛋白标准物质候选物的制备与纯化（英文）

随着转基因技术的迅猛发展,转基因产品的安全性受到了广泛关注。转基因检测用有证标准物质在确保转基因产品定性、定量检测结果的可比性和可追溯性方面发挥着重要作用。但转基因蛋白质标准物质的开发相对缓慢,其中一个难点是制备高纯度的转基因蛋白质候选物。苏云金芽胞杆菌Bacillus thuringiensis cry1Ah1基因因其对亚洲玉米螟等鳞翅目害虫有很好的杀虫活性,已用于转基因抗虫作物的研制,并获得具有较好抗虫性状的转基因株系。为了研发Cry1Ah蛋白有证标准物质,亟需建立其制备及纯化体系。文中优化了利用Bt表达系统制备Cry1Ah蛋白的体系,利用离子交换色谱法和排阻色谱法逐级纯化的方法,获得了高纯度的Cry1Ah蛋白(排阻色谱纯度:99.6%)。生物活性测定结果表明,纯化的Cry1Ah蛋白与原毒素对小菜蛾Plutella xylostella的杀虫活性没有显著差异。最后使用Edman降解法和质谱法确定了Cry1Ah蛋白活化后的氨基酸序列。综上所述,获得的Cry1Ah纯蛋白可用于蛋白质标准物质的研制。     

一种新的Bt杀虫基因HJC原核表达蛋白活性的鉴定

HJC基因是由2个Bt基因(cry1Ab和vip3)经过人工融合而成,具有更广谱的杀虫活性,可延缓害虫产生交互抗性的时间。将已构建好的携带HJC基因的重组质粒pET28a-HJC转化到大肠杆菌BL21中诱导表达。该HJC融合蛋白主要以包涵体形式存在,变性条件下使用镍亲和层析柱对其进行纯化,并经尿素梯度透析复性后,进行免疫反应活性及美国白蛾杀虫活性测定。Western blot结果显示,该原核表达蛋白与转HJC基因水稻中的HJC蛋白有相同的免疫反应性,对美国白蛾也有一定的杀虫活性,可以替代植物外源蛋白进行转HJC基因产品的食用安全性评价。     

Insecticidal peptides from the theraposid spider Brachypelma albiceps: An NMR-based model of Ba2

Soluble venom and purified fractions of the theraposid spider Brachypelma albiceps were screened for insecticidal peptides based on toxicity to crickets. Two insecticidal peptides, named Ba1 and Ba2, were obtained after the soluble venom was separated by high performance liquid chromatography and cation exchange chromatography. The two insecticidal peptides contain 39 amino acid residues and three disulfide bonds, and based on their amino acid sequence, they are highly identical to the insecticidal peptides from the theraposid spiders Aphonopelma sp. from the USA and Haplopelma huwenum from China indicating a relationship among these genera. Although Ba1 and Ba2 were not able to modify currents in insect and vertebrate cloned voltage-gated sodium ion channels, they have noteworthy insecticidal activities compared to classical arachnid insecticidal toxins indicating that they might target unknown receptors in insect species. The most abundant insecticidal peptide Ba2 was submitted to NMR spectroscopy to determine its 3-D structure; a remarkable characteristic of Ba2 is a cluster of basic residues, which might be important for receptor recognition.     

Large scale production of Bacillus thuringiensis PS149B1 insecticidal proteins Cry34Ab1 and Cry35Ab1 from Pseudomonas fluorescens

The 14kDa (Cry34Ab1) and 44kDa (Cry35Ab1) binary insecticidal proteins are produced naturally by Bacillus thuringiensis PS149B1 as parasporal inclusion bodies. Here, we show production of these two insecticidal proteins in recombinant Pseudomonas fluorescens and their subsequent purification to near homogeneity to provide large quantities of protein for safety-assessment studies associated with the registration of transgenic corn plants. The gene sequence specific for each protein was expressed in P. fluorescens and fermented at the 75-L scale. For Cry34Ab1, the protein accumulated as insoluble inclusion bodies, and was purified by extraction directly from the cell pastes at pH 3.4 with a sodium acetate buffer, selective precipitation at pH 7.0, and differential centrifugation. For Cry35Ab1, the protein was extracted from the purified inclusion bodies with sodium acetate buffer (pH 3.5) containing 0.5M urea, followed by diafiltration. No chromatography steps were required to produce over 30g of lyophilized protein powder with purity greater than 98%, while retaining full insecticidal activity against Western corn rootworm larvae. The proteins were further characterized to assure identity and suitability for use in safety-assessment studies.     

Mechanisms of the insecticidal action of TEL (Talisia esculenta lectin) against Callosobruchus maculatus (Coleoptera: Bruchidae)

Plant lectins have insecticidal activity that is probably mediated through their ability to bind carbohydrates. To examine the influence of sugars on the insecticidal activity of a lectin from Talisia esculenta seeds (TEL), the lectin was mixed with mannose, glucose, or mannose plus glucose. Mannose abolished the insecticidal activity. Affinity chromatography showed that TEL bound to midgut proteins of the insect Callosobruchus maculatus. Immunoblotting showed that TEL recognized some proteins, probably glycoproteins, present in the midgut membrane of this insect. The principal proteases responsible for digestive proteolysis in fourth instar larvae of C. maculatus were purified by chromatography on activated thiol-Sepharose. These purified proteases were unable to digest TEL after a 15-h incubation. These results suggest that the insecticidal activity of TEL involves a specific carbohydrate-lectin interaction with glycoconjugates on the surface of digestive tract epithelial cells, as well as binding to assimilatory glycoproteins present in midgut extracts and resistance to enzymatic digestion by cysteine proteinases.     

鳞翅目昆虫中肠Bt杀虫蛋白受体研究进展

杀虫晶体蛋白(insecticidal crystal proteins,ICPs;含有Cry和Cyt 2大家族)和营养期杀虫蛋白(vegetative insecticidal proteins,Vips)等Bt杀虫蛋白可有效防治鳞翅目害虫,其中Cry应用最广泛。然而,一些地区的鳞翅目害虫已对Bt杀虫蛋白产生了抗性。目前,普遍认为鳞翅目昆虫中肠受体与Bt杀虫蛋白结合能力的改变是导致其对Bt杀虫蛋白产生抗性的最主要因素。在鳞翅目昆虫中,Cry受体是研究得最为透彻的Bt受体,已经被证实的有氨肽酶N、钙黏蛋白、碱性磷酸酶和ABC转运蛋白等。Vips杀虫蛋白类与鳞翅目昆虫中肠受体的结合方式与Cry杀虫蛋白相似,但结合位点与Cry杀虫蛋白不同。本文从结构特点、作用机制及不同鳞翅目昆虫间的表达差异等角度对以上4种鳞翅目昆虫中肠Bt受体进行了综述,并提出如下展望:(1)以棉铃虫或小菜蛾等鳞翅目昆虫为农业害虫模式生物进行深入研究,阐明其对Bt杀虫蛋白产生抗性的机制,为研究其他鳞翅目农业害虫对Bt杀虫蛋白产生抗性的机制提供理论借鉴;(2)鉴于在不同鳞翅目昆虫间,中肠Bt受体与Bt杀虫蛋白结合存在差异,且同一Bt杀虫蛋白与鳞翅目昆虫Bt受体并不专一性结合,Bt杀虫蛋白多基因组合策略是较为有效的田间鳞翅目昆虫防治策略,是今后一段时间内Bt杀虫蛋白应用的发展方向。     

Brush border membrane aminopeptidase-n in the midgut of the gypsy moth serves as the receptor for the CryIA(c) δ-endotoxin of Bacillus thuringiensis

Aminopeptidase-N (AP-N) was purified from gypsy moth (Lymantria dispar, L.) brush border membrane vesicles (BBMV) proteins by mono-Q chromatography and Superdex-75 gel filtration in the presence of the zwitterionic detergent, CHAPS, using FPLC. The purified AP-N, identified by its enzymatic activity, had an apparent size of 100 kDa, and was identified as the unique Bacillus thuringiensis insecticidal toxin, CryIA(c), binding protein. AP-N clearly displayed strong binding to CryIA(c), exhibiting little or no binding to CryIA(a) or CryIA(b), and showing no binding for the coleopteran-specific toxin, CryIIIA. Protein blots of the BBMV proteins probed with biotin-labeled and ¹²⁵I-labeled insecticidal proteins revealed that CryIAc binds only to 120 kDa protein which is a slightly larger size in comparison to purified AP-N. Antibodies raised against the gypsy moth AP-N demonstrated that the purified AP-N and the 120 kDa CryIA(c) binding protein of total BBMV proteins are antigenically identical.