我国仓贮昆虫病原芽孢杆菌的研究

对９５１个样品分离鉴定,有７４７个样品含芽孢杆菌,有菌率为７８．５５％．共分离得到芽孢杆菌１１３８株,其中苏云金杆菌（Ｂａｃｉｌｌｕｓｔｈｕｒｉｎｇｉｅｎｓｉｓ,简称Ｂ．ｔ）１４３株,占１２．５％;球形芽孢杆菌（Ｂａｃｉｌｌｕｓｓｐｈａｅｒｉｃｕｓ,简称Ｂ．ｓ）１１株,占０．９７％;其他芽孢杆菌９８４株,占８６．４０％．从芽孢杆菌中选出产生晶体、苏云金素或磷酸酯酶Ｃ（ＰｈｏｓｐｈａｌｉｐａｓｅＣ,简称ＰＬＣ）的毒素菌株１６８株,其中Ｂ．ｔ占１４３株,Ｂ．ｓ有５株,其他芽孢杆菌１０株．在产毒素菌株中,经测定有１２０株菌对供试昆虫毒性达标．占７７．９２％．不同菌株的杀虫毒素、杀虫范围和毒力各异,认为这种差异取决于毒素和虫种两方面的特异性．     

芽孢杆菌原生质体融合技术

微生物原生质体融合技术是近20年来国内外细胞工程领域的一个研究热点。1972年匈牙利学者Ｆｅｒｅｎｃｚｙ率先进行了微生物原生质体融合的研究[1]。在1976年匈牙利学者Ｆｏｌｄｅｒ和Ａｌｆｏｌｄ则首次报道了用ＰＥＧ或新生态磷酸钙诱导巨大芽孢杆菌(Ｂａｃｉｌｌｕｓｍｅｇａｔｅｒｉｕｍ)种内株间原生质体融合[2];同年法国的Ｓｃｈａｅｆｆｅｒ等也用ＰＥＧ诱导枯草芽孢杆菌(Ｂ.ｓｕｂｔｉｌｉｓ)进行种内株间原生质体融合获得成功[3]。有关芽孢杆菌原生质体融合的研究,在国内直至1981年才见报道[4]。经典改变微生物遗传性状的手段有两…     

苏云金杆菌遗传工程杀虫剂

苏云金芽孢杆菌（Ｂａｃｉｌｌｕｓ ｔｈｕｒｉｎｇｉｅｎｓｉｓ,简称 Ｂｔ）是一种对昆虫有致病作用的细菌。其杀虫活性物质主要是杀虫晶体蛋白质（Ｉｎｓｅｃｔｉｃｉｄｅ ｃｒｙｓｔａｌ ｐｒｏｔｅｉｎ,ＩＣＰ）。由于该杀虫剂对人、畜无害,无致癌和致畸作用,是一种有着广泛应用前景的微生物杀虫剂。 根据１９９８年最新的分类,把ＩＣＰ基因分为２５类ｃｒｙ基因和２类ｃｙｔ基因,每类ＩＣＰ基因产生针对不同昆虫的晶体蛋白质。例如,ｃｒｙ３Ａ基因产生杀鞘翅目昆虫的晶体蛋白质,而ｃｒｙ１基因所产生的活性物质主要用于防治鳞…     

化感物质对枯草芽孢杆菌（Bacillus subtilis）在厌氧条件下和生长？…

研究由秸秆腐解产生的化感物质：阿魏酸（ｔ－ＦＡ）、对羟基苯甲酸（ｐ－ＨＡ）和苯甲酸（ＢＡ）在不同浓度下对厌氧培养的枯草芽孢杆菌（Ｂａｃｉｌｌｕｓ ｓｕｂｔｉｌｉｓ）的生长及其反硝化活性的影响。结果表明,３种浓度的阿魏酸（５．１５、２．５８、０．２６ｍｍｏｌ／Ｌ）均表现出对枯草芽孢杆菌的生长有抑制作用。对羟基苯甲酸（０;．３６、３．６２、７．２４ｍｍｏｌ／Ｌ）对生长影响不明显。８．１９ｍｍｏｌ／Ｌ和     

一种球形芽孢杆菌的选择性培养基

一种球形芽孢杆菌的选择性培养基袁志明,陈宗胜,刘娥英,张用梅,蔡全信（中国科学院武汉病毒研究所,武汉４３００７１）球形芽孢杆菌（ＢａｃｉｌｌｕｓｓｐｈａｅｒｉｃｕｓＮｅｉｄｅ）是一种孢子囊末端膨大、芽孢球形的好氧性芽孢杆菌。其中ＤＮＡ同源型ⅡＡ型,血...     

苏云金芽孢杆菌的电穿孔及其工程菌的构建

本文对苏芸金芽孢杆菌 (Ｂａｃｉｌｌｕｓｔｈｕｒｉｎｇｉｅｎｓｉｓ,Ｂｔ)和蜡状芽孢杆菌 (Ｂａｃｉｌｌｕｓｃｅｒｅｕｓ,Ｂｃ)等部分菌株的电穿孔转化进行了研究 ,主要从电穿孔的供体质粒和受体菌株等方面讨论了各种因素对该方法的影响。同时利用电穿孔方法对部分Ｂｔ的高效野生株进行遗传改良 ,希望获得含有ｃｒｙ1Ａｃ和ｃｒｙ1Ｃ高效基因组合的对鳞翅目棉铃虫、甜菜夜蛾都有效的广谱工程株。利用质粒 ｐＳＢ1 40 2 (ｃｒｙ1Ｃ) ,ｐＡＭＹ(ｃｒｙ1Ａｃ) ,ｐＮＱ1 2 2 (Ｃｍｒ)对所有的供试菌株进行电穿孔转化 ,不同菌株表现出不同的转…     

蛋白酶对解淀粉芽孢杆菌α－淀粉酶活力的影响

目前工业生产α－淀粉酶的主要菌种是解淀粉芽孢杆菌（Ｂａｃｉｌｌｕｓａｍｙｌｏｌｉｑｕｅｆａｃｉｅｎｓ）,该菌在培养条件下不但产生α－淀粉酶,同时也产生一定比例的蛋白酶。本文研究了不同来源的蛋白酶对解淀粉芽孢杆菌ＢＦ７６５８和８６３１５α－淀粉酶活性的影响,结果发现中性蛋白酶对两种α－淀粉酶活性无显著影响,而２７０９碱性蛋白酶能使α－淀粉酶活性丧失６０％以上。     

二种重组α-乙酰乳酸脱羧酶在啤酒生产中降低双乙酰的作用

利用已构建的含有短芽孢杆菌（Ｂａｃｉｌｌｕｓ ｂｒｅｖｉｓ）和产气肠杆菌（Ｅｎｔｅｒｏｂａｃｔｅｒ　ａｅｒｏｇｅｎｅｓ）α－乙酰乳酸脱羧酶（α－ａｃｅｔｏｌａｃｔａｔｅ ｄｅｃａｒｂｏｘｙｌａｓｅ, α-ＡＬＤＣ）基因的工程菌株,并使它们分别在大肠杆菌中高效表达,获得重组α－ＡＬＤＣ。在实验室,用 ２Ｌ体积的麦芽汁进行啤酒生产试验,添加 ２种重组α－ＡＬＤＣ后,使啤酒中的双乙酰含量快速下降到或始终保持在０.１ｍｇ／Ｌ以下。证明得到的重组ａ－ＡＬＤＣ能有效地降低啤酒中双乙酰含量。     

固氮菌接种对棉苗氮磷钾吸收,某些酶活性及产量的影响

Ｓｐｅｕｍｏｓｐｈｅｒｅ ｍｏｄｅｌｓ和盆栽试验结果表明,海岛棉（Ｇｏｓｓｙｐｉｕｍ ｂａｒｂａｄｅｎｓｅ Ｌ．）苗接种自生固氮菌（Ａｚｏｔｏｂａｃｔｅｒ ｓｐ．）、巴西固氮螺菌ＮＯ４０（Ａｚｏｓｐｉｒｉｌｌｕｍ ｂｒａｓｉｌｅｎｓｅ ＮＯ４０）、多粘芽孢杆菌（Ｂａｃｉｌｌｕｓ ｐｏｌｙｍｙｘａ ＣＦ）和根瘤菌（Ｒｈｉｚｏｂｉｕｍ）,和以自生固氮菌分别与其它３种供试菌种两者混合菌,能增强棉花根际固     

二种重组α-乙酰乳酸脱羧酶在啤酒生产中降低双乙酰的作用

利用已构建的含有短芽孢杆菌（Ｂａｃｉｌｌｕｓ ｂｒｅｖｉｓ）和产气肠杆菌（Ｅｎｔｅｒｏｂａｃｔｅｒ　ａｅｒｏｇｅｎｅｓ）α－乙酰乳酸脱羧酶（α－ａｃｅｔｏｌａｃｔａｔｅ ｄｅｃａｒｂｏｘｙｌａｓｅ, α-ＡＬＤＣ）基因的工程菌株,并使它们分别在大肠杆菌中高效表达,获得重组α－ＡＬＤＣ。在实验室,用 ２Ｌ体积的麦芽汁进行啤酒生产试验,添加 ２种重组α－ＡＬＤＣ后,使啤酒中的双乙酰含量快速下降到或始终保持在０.１ｍｇ／Ｌ以下。证明得到的重组ａ－ＡＬＤＣ能有效地降低啤酒中双乙酰含量。     

九香虫血淋巴及其纯化蛋白抑菌活性的研究

对九香虫AsporgopuschinensisDallas血淋巴及其血淋巴蛋白质分离物的抗菌活性进行了研究,抗菌活性检测指示菌为大肠杆菌Escherichiacoli和金黄色葡萄球菌Staphilocalliesacereus。测定结果表明,九香虫血淋巴及其离心上清液都具有明显的抗菌活性。用凝胶过滤法从血淋巴蛋白分离提纯获得一种小分子肽,SDS PAGE电泳为单一带,分子量约为1～1 4 4kD。该小分子蛋白对大肠杆菌和金黄色葡萄球菌都有抑菌作用,与血淋巴对2种细菌的抗菌性一致,表明其是九香虫血淋巴中具抗菌作用的主要物质之一。     

蜚蠊灭菌肽的诱导及初步分离分析

昆虫经诱导盾产生灭菌肽的研究近年来已有很大进展,有关这方面的研究工作绝大多数都是以有翅亚纲内生翅类鳞翅目(主要是蚕类)昆虫和少数双翅目昆虫为材料.本文首次以有翅亚纲外生翅类蜚蠊目的美洲蜚蠊(Ptriplaneta americana L.)为实验昆虫,用Escherichia coil K₁₂ strain D₃₁作诱导源,对不同发育期、不同性别、不同成虫期的蜚蠊进行诱导后,采用含菌培养基平板测活方法,就存在个体数及能产生抗菌物质的个体数进行了初步的研究.发现成虫日龄在10天之内的雄性蜚蠊能够产生抗菌物质的个体百分比最高.抗菌物质出现的高峰期是在诱导后第三、四天.用滴滴涕和溴氰菊酯作诱导源对雄性蜚蠊的诱导实验表明,杀虫剂也能诱导蜚蠊产生抗菌物质,而且所诱导产生抗菌物质的活性强度(用抑菌圈直径表示)高于大肠杆菌所诱导的.滴滴涕和溴氰菊酯的重复诱导可提高蜚蠊产生抗菌物质个体百分比.蜚蠊经诱导后产生的抗菌物质具有广谱性,对苏云金杆菌、金黄色葡萄球菌、枯草杆菌及绿脓杆菌等有较强的抗菌活性,而对大肠杆菌D₃₁、大肠杆菌、粘质沙雷氏杆菌和溶壁微球菌等有较弱的抗菌活性.用肽类物质的指纹图谱法分离蜚蠊血淋巴抗菌物质,发现经诱导后血淋巴中确有新的肽类物质产生,该物质具抗菌活性,用DABITC法分析,其N-末端氨基酸为赖氨酸.     

柞蚕滞育蛹的体液防卫反应

本文对柞蚕Antheraea pernyi滞育蛹体液防卫的某些特性做了报道。结果表明：(1)经注射大肠杆菌诱导6h后,体液中出现了抗菌活性物质。诱导7d后,抗菌活力达到峰值,15d后活力消失。用生理盐水诱导的反应较快,4d后达到峰值,10d后活力消失。(2)经大肠杆菌诱导后的体液对苏芸金杆菌、大肠杆菌、枯草杆菌及金黄葡萄球菌均有杀菌效应。在最初5min内杀菌效率较高。注射生理盐水诱导的体液较大肠杆菌诱导的杀菌效果低。(3)用昆虫病原菌苏芸金杆菌及非病原菌大肠杆菌作不同的诱导源,诱导1d后的体液对大肠杆菌的抗菌活力差别不显著,但对柞蚕蛹的生活力及发育有明显影响;用苏芸金杆菌诱导的柞蚕滞育蛹3d后死亡,用大肠杆菌诱导的蛹能正常羽化。(4)在一定范围内用不同剂量的大肠杆菌进行诱导,其抗菌活力不受影响,但超过一定限度,既使是非病原菌也可以突破昆虫的防卫功能。     

注射大肠杆菌或超声波诱导家蚕及蓖麻蚕产生抗菌物质的比较研究

本文报道:1.蓖麻蚕hilosamia cynthia ricini、家蚕Bombyx mori及柞蚕Antheraea pernyi注射大肠杆菌Escherichia coli或超声波处理均能诱导血淋巴产生抗菌物质。同一蚕种诱导产物相同,不同蚕种间差异明显。2.家蚕不同发育阶段的个体诱导产生的抗菌物质基本相同;不同性别的家蚕蛹的诱导产物的相对量有差异;不同品种家蚕蛹对诱导的应答潜伏期也不尽相同。3.注射聚肌胞核苷酸(PolyI:C)诱导家蚕血淋巴产生一种明显地不同于其它诱导源诱导的抗菌物质,此物质在酸性聚丙烯酰胺凝胶电泳中泳动速度较快。 本研究的所有结果均表明,诱导绢丝昆虫产生抗菌物质的诱导源是非专一性的,即诱导源与诱导产物之间无对应的关系。     

Exoproteinases of the type A in pathogenesis of insect bacterial diseases

Immune inhibitors produced in infected larvae of Galleria mellonella by such entomopathogens as Pseudomonas aeruginosa, Serratia marcescens and Heterorhabditis bacteriophora effectively blocked in vitro bactericidal activity of insect haemolymph against Escherichia coli D31, both in Galleria mellonella and Pieris brassicae pupae previously vaccinated with Enterobacter cloacae. Even at a trace concentration, the extracellular proteinases, by proteolytic degradation, totally destroyed the activity of cecropin peptides from Galleria and cecropin-like and attacin-family proteins from Pieris, but no ability to destroy antibacterial activity was shown by extracts obtained from Galleria larvae killed by massive doses of bacterial saprophytes. It is suggested that by blocking antibacterial immune response of the host, the proteinases help the bacteria to multiply in the haemolymph, thus they could be considered an important factor in the pathogenesis of bacterial diseases of insects.     

Acaloleptins A: inducible antibacterial peptides from larvae of the beetle, Acalolepta luxuriosa

We purified and characterized three structurally related antibacterial peptides with a molecular mass of 8 kDa (acaloleptins A1, A2, and A3) from the hemolymph of immunized larvae of the Udo longicorn beetle, Acalolepta luxuriosa. These peptides have the same 6 N-terminal amino acid residues and show potent antibacterial activity against some Gram-negative bacteria. The three peptides are thought to be isoforms. Reverse phase HPLC analysis of the hemolymph of immunized and naive larvae showed that acaloleptins A1, A2, and A3 were inducible and suggested that all three peptides were produced in a single insect. We determined the complete amino acid sequence of acaloleptin A1: Acaloleptin A1 consists of 71 amino acid residues and shares significant sequence similarity with coleoptericin and holotricin 2, which were isolated from other coleopteran insects. Furthermore, the 29 C-terminal residues of acaloleptin A1 had 40% identity with the 30 C-terminal residues of hymenoptaecin found in honeybees. Arch. Insect Biochem.     

Comparison of parasitism by Cotesia glomerata with bacterial infection and wounding in Pieris brassicae: induction of new haemolymph polypeptides and changes in humoral immune response

In Pieris brassicae, parasitism by Cotesia glomerata and bacterial infection are differentiated with respect to haemolymph protein arrays, and production or suppression of antibacterial agents. Bacteriolytic activity in haemolymph from parasitized larvae was slightly, but significantly, higher 24h post-treatment than that of untreated and wounded controls. Micrococcus lysodeikticus- or lipopolysaccharide-(LPS) injected insects exhibited an 11-fold greater response than those parasitized. At 24h post-treatment, antibacterial activity against Escherichia coli was observed in haemolymph from all but untreated larvae. Injection of Grace's medium, M. lysodeikticus or LPS, caused a greater than threefold response than parasitization or wounding. The protein banding patterns of parasitized hosts did not correspond to those of the other treatments. Two parasitoid-induced proteins (38 and 128 kDa) were examined. Both were found in parasitized insects, not in those wounded, injected with Grace's medium, M. lysodeikticus or LPS. Neither protein was bacteriolytic or bacteriostatic in inhibition zone assays.     

Insect immunity. Purification and characterization of a family of novel inducible antibacterial proteins from immunized larvae of the dipteran Phormia terranovae and complete amino-acid sequence of the predominant member, diptericin A

Injury or injection of live bacteria into third instar larvae of the dipteran insect Phormia terranovae results in the appearance in the haemolymph of at least five groups of heat-stable, more or less basic peptides with antibacterial activity against Escherichia coli. Three of these peptides have been purified. The amino acid sequence has been completely established for one of these and partially (first 40 residues from the N-terminus) for the two others. The sequences show marked homologies indicating that the three peptides belong to a common family. They are not related to other known antibacterial peptides from insects [lysozymes, cecropins (including sarcotoxin I) and attacins]. We propose the name of diptericins for this new family of antibiotic molecules.     

The sandfly Lutzomyia longipalpis shows specific humoral responses to bacterial challenge

Abstract The presence of immune molecules induced by microorganisms in the haemolymph of Lutzomyia longipalpis sandflies has been investigated. Injections of Escherichia coli and Micrococcus luteus into female sandflies induced anti-bacterial activity in the haemolymph. Inhibition zone assays showed that haemolymph from E.coli and M.luteus injected sandflies differentially inhibited M.luteus growth. This differential effect was specific to M. luteus infection since anti-Zs. coli activity was similar in haemolymph from both E.coli oxM.luteus injected sandflies. Haemolymph following injection of either bacteria showed the induction of a 4kDa peptide. Haemolymph from M.luteus injected sandflies also contained a 33 kDa polypeptide which was absent in haemolymph from E.coli and control uninfected insects. Sandflies, in common with other insects, were shown to possess general and specific humoral immune responses to the presence of microorganisms.     

Antibacterial activity in the hemolymph of myriapods (Arthropoda)

The hemolymphs of two diplopod (Chicobolus sp. and Rhapidostreptus virgator) and two chilopod species (Lithobius forficatus and Scolopendra cingulata) were tested for the presence of antibacterial substances using Petri dish tests. The native hemolymph of all species had substances acting on living Micrococcus luteus, whereas only Rhapidostreptus, Scolopendra, and Lithobius were effective against lyophilized Micrococcus. The antibacterial activity against living Micrococcus increased after inoculation with bacteria (Enterobacter cloacae beta-12) in Chicobolus and Rhapidostreptus and also against lyophilized Micrococcus in the latter. Thus, these effects appear to be inducible. None of the myriapods tested had any bacteriostatic effect on Escherichia coli D-31 whereas the growth of gram-negative E. cloacae was inhibited. The antibacterial substances in the diplopod species were unstable when heated but were resistant to freezing. At least two antibacterial substances (a lysozyme-like one and another substance) are considered to occur in Myriapoda.