嗜线虫致病杆菌血腔毒素Tp40对大蜡螟幼虫体内酶活和中肠组织的影响

嗜线虫致病杆菌Xenorhabdus nematophila在侵入到寄主昆虫血腔后能够成功地逃避或抑制寄主昆虫的免疫反应并快速杀死昆虫。为深入了解嗜线虫致病杆菌的杀虫机理,明确关键的致病因子,作者应用盐析和制备型非变性凝胶电泳等方法,从嗜线虫致病杆菌HB310菌株的细胞内分离纯化了一种新的杀虫蛋白——Tp40,该蛋白对大蜡螟Galleria mellonella具有高血腔注射活性,对大蜡螟5龄幼虫的LD₅₀为68.54 ng/头。本文检测了该毒素对大蜡螟幼虫的致病特性,注射Tp40毒素后,大蜡螟幼虫表现出兴奋和痉挛等症状,当以不低于（70±0.02）ng/头的剂量注射Tp40,大蜡螟幼虫均在20 min内死亡,但试虫的体色 、血淋巴的颜色以及血细胞的形态没有发生明显的变化。对大蜡螟体内酶活性的测定结果显示,在注射LD₅₀剂量的Tp40蛋白后,试虫体内羧酸酯酶和乙酰胆碱酯酶活力都明显的高于对照（P<0.05）,而酚氧化酶活力显著低于对照（P<0.05）。对大蜡螟幼虫中肠的组织病理学研究显示：这种42 kDa蛋白能够破坏试虫的中肠组织,导致其肠壁细胞出现排列紊乱、脱落和围食膜消失。据此推测,Tp40与嗜线虫致病杆菌对寄主昆虫的免疫抑制有关,寄主中肠组织可能是其作用靶标之一。     

嗜线虫致病杆菌HB310菌株杀虫蛋白的纯化及活性鉴定

嗜线虫致病杆菌Xenorhabdus nematophila HB310是从河北省土壤中筛选出的一株昆虫病原线虫体内分离纯化获得的共生菌,该菌的发酵液对多种昆虫有较高的杀虫活性。利用85％饱和度的硫酸铵盐析分别获得胞内蛋白提取物和上清液中胞外蛋白提取物,生测结果表明这两种蛋白提取物中都含有胃毒素和血腔毒素。通过制备型非变性凝胶电泳对蛋白提取物进行分离和纯化,得到了3种有杀虫活性的毒素蛋白（毒素Ⅰ、毒素Ⅱ和毒素Ⅲ）,胞内的毒素蛋白与分泌到胞外上清液中的毒素蛋白是同种蛋白。毒素Ⅰ和毒素Ⅱ对棉铃虫初孵幼虫有明显的胃毒活性,但没有血腔毒性;毒素Ⅲ对大蜡螟幼虫有很强的血腔毒性,LD₅₀为0.18 μg/头。SDS-PAGE图谱显示毒素Ⅰ和毒素Ⅱ是由多个多肽组成的复合蛋白,而毒素Ⅲ只分离出一条多肽。毒素Ⅱ在50℃处理10 min,其杀虫活性没有显著变化;70℃处理10 min对毒素Ⅲ杀虫活性没有显著影响。     

昆虫病原线虫共生菌Tc毒素研究进展

来自昆虫病原线虫共生菌的Tc毒素是一类多亚基组成的高分子蛋白复合物,对多种农业害虫具有广谱的胃毒活性,这类毒素与目前已知的其它杀虫蛋白同源性非常低,有可能成为新的杀虫资源。本文对来自昆虫病原线虫共生菌的Tc毒素的特性、作用模式等方面的国内外研究进展进行了综述。     

昆虫病原线虫共生细菌致病机理的研究进展

昆虫病原线虫共生细菌是寄生于昆虫病原线虫肠道内的一种细菌,革兰氏染色呈阴性,属肠杆菌科(Ｅｎｔｅｒｏｂａｃｔｅｒｉａｃｅａｅ)细菌[1]。它包含两个属———嗜线虫致病杆菌属(Ｘｅｎｏｒｈａｂｄｕｓ)和发光杆菌属(Ｐｈｏｔｏｒｈａｂｄｕｓ),它们分别与斯氏线虫(Ｓｔｅｉｎｅｒｎｅｍａ)和异小杆线虫(Ｈｅｔｅｒｏｒｈａｂｄｉｔｉｓ)共生。这两种线虫由于杀虫能力强,是最有应用潜力的昆虫病原线虫。它们之间的共生关系可以概括为:共生菌存在于线虫的肠道内,线虫携带共生菌进入寄主昆虫体内,并将共生菌释放到昆虫的血腔中;共生菌在昆…     

昆虫病原线虫共生菌杀虫蛋白的研究进展

昆虫病原线虫共生菌杀虫蛋白对许多农业害虫都有很明显的毒杀作用,作为一种新型的具有开发潜力和应用前景的生物防治资源,已经引起科学家们的关注并成为国内外研究的热点之一.综述了昆虫病原线虫共生菌杀虫蛋白基因的结构和功能及其杀虫机制方面的研究进展.     

昆虫病原线虫共生细菌的代谢产物

昆虫病原线虫共生细菌寄生于昆虫病原线虫肠道内 ,二者互惠共生。该菌革兰氏染色阴性 ,属肠杆菌科 (Ｅｎｔｅｒｏｂａｃｔｅｒｉａｃｅａｅ) ,包含两个属———嗜线虫致病杆菌属 (Ｘｅｎｏｒｈａｂｄｕｓ)和发光杆菌属 (Ｐｈｏｔｏｒｈａｂｄｕｓ)。其中嗜线虫致病杆菌与斯氏线虫 (Ｓｔｅｉｎｅｒｎｅｍａ)共生 ,发光杆菌与异小杆线虫 (Ｈｅｔｅｒｏｒｈａｄｉｔｉｓ)共生。近几年 ,随着对共生菌研究的逐渐深入 ,发现共生菌能产生许多有应用潜力的代谢产物 ,如杀虫蛋白、抑菌物质、抗癌物质、胞外酶、胞内晶体蛋白、色素及荧光素等。特别是…     

致病杆菌属和光杆状菌属细菌杀虫毒素蛋白

致病杆菌属和光杆状菌属细菌是一类分别与斯氏线虫属和异小杆属线虫共生的昆虫病原细菌 ,属肠杆菌科 ,此类细菌产生的杀虫毒素蛋白是近年来发现的一类高效、杀虫谱广的新型杀虫蛋白。此类毒素蛋白对多种昆虫具有注射和口服毒性 ,在同一菌株中有多个杀虫基因 ,各杀虫蛋白基因之间具有协同毒力效应 ,杀虫蛋白基因在大肠杆菌和植物中表达的毒素蛋白对多种害虫具有口服毒性。     

伯氏致病杆菌IDP16 蛋白抑制大蜡螟的免疫反应

[目的]从伯氏致病杆菌(Xenorhabdus bovienii)胞外组分中分离纯化出能够抑制大蜡螟(Galleria mellonella)免疫反应的一种蛋白,研究其在昆虫病原线虫及其共生菌致病过程中的作用.[方法]采用硫酸铵沉淀和柱层析的方法对活性蛋白进行分离和纯化,通过体内注射并观察血淋巴黑化进行活性蛋白的筛选;采用荧光微球和琼脂糖小球评价活性蛋白对血细胞吞噬、包被作用的影响;采用双向电泳结合质谱分析对活性蛋白进行鉴定,设计引物用PCR的方法克隆其编码基因,利用pET 30a载体进行原核表达,以亲和层析纯化重组蛋白.[结果]纯化得到一个昆虫免疫抑制蛋白,命名为IDP16,该蛋白可显著抑制大蜡螟血淋巴中的多酚氧化酶活性,降低血细胞的吞噬和包被作用.克隆得到其编码基因并进行了原核表达,重组蛋白仍具有免疫抑制活性.[结论]伯氏致病杆菌产生的IDP16蛋白能够抑制昆虫的免疫反应,在共生菌和宿主昆虫互作过程中起着重要的作用.     

昆虫病原线虫共生菌杀虫毒素研究进展

对昆虫病原线虫共生菌杀虫毒素的种类、与口服毒性有关的杀虫毒素以及口服毒性与杀虫毒素基因的关系等研究进展进行了综述,并对未来的研究方向提出了作者的见解。     

昆虫病原线虫共生细菌共生性的分子生物学研究进展

嗜线虫致病杆菌属Xenorhabdus和发光杆菌属Photorhabdus细菌隶属肠杆菌科Enterobacteriaceae,对多种害虫致病能力强,分别与斯氏属Steinernema和异小杆属Heterorhabditis昆虫病原线虫互惠共生。该两属共生细菌既存在对昆虫寄主的病原性,又存在与线虫寄主的共生性。共生细菌与其线虫寄主的共生性主要表现以下4方面：（1）细菌产生食物信号诱导滞育不取食的感染期线虫恢复;（2）细菌为线虫生长与繁殖提供营养;（3）细菌能于感染期线虫的肠道定殖与生长;（4）细菌产生杀线虫毒素杀死非共生线虫。本文综述了共生菌以上4方面的共生性及其相关的分子机制。     

A novel secreted protein toxin from the insect pathogenic bacterium Xenorhabdus nematophila

The bacterium Xenorhabdus nematophila is an insect pathogen that produces several proteins that enable it to kill insects. Screening of a cosmid library constructed from X. nematophila strain A24 identified a gene that encoded a novel protein that was toxic to insects. The 42-kDa protein encoded by the toxin gene was expressed and purified from a recombinant system, and was shown to kill the larvae of insects such as Galleria mellonella and Helicoverpa armigera when injected at doses of around 30-40 ng/g larvae. Sequencing and bioinformatic analysis suggested that the toxin was a novel protein, and that it was likely to be part of a genomic island involved in pathogenicity. When the native bacteria were grown under laboratory conditions, a soluble form of the 42-kDa toxin was secreted only by bacteria in the phase II state. Preliminary histological analysis of larvae injected with recombinant protein suggested that the toxin primarily acted on the midgut of the insect. Finally, some of the common strategies used by the bacterial pathogens of insects, animals, and plants are discussed.     

Insecticidal pilin subunit from the insect pathogen Xenorhabdus nematophila

Xenorhabdus nematophila is an insect pathogen and produces protein toxins which kill the larval host. Previously, we characterized an orally toxic, large, outer membrane-associated protein complex from the culture medium of X. nematophila. Here, we describe the cloning, expression, and characterization of a 17-kDa pilin subunit of X. nematophila isolated from that protein complex. The gene was amplified by PCR, cloned, and expressed in Escherichia coli. The recombinant protein was refolded in vitro in the absence of its cognate chaperone by using a urea gradient. The protein oligomerized during in vitro refolding, forming multimers. Point mutations in the conserved N-terminal residues of the pilin protein greatly destabilized its oligomeric organization, demonstrating the importance of the N terminus in refolding and oligomerization of the pilin subunit by donor strand complementation. The recombinant protein was cytotoxic to cultured Helicoverpa armigera larval hemocytes, causing agglutination and subsequent release of the cytoplasmic enzyme lactate dehydrogenase. The agglutination of larval cells by the 17-kDa protein was inhibited by several sugar derivatives. The biological activity of the purified recombinant protein indicated that it has a conformation similar to that of the native protein. The 17-kDa pilin subunit was found to be orally toxic to fourth- or fifth-instar larvae of an important crop pest, H. armigera, causing extensive damage to the midgut epithelial membrane. To our knowledge, this is first report describing an insecticidal pilin subunit of a bacterium.     

Txp40, a ubiquitous insecticidal toxin protein from Xenorhabdus and Photorhabdus bacteria

Xenorhabdus and Photorhabdus are gram-negative bacteria that produce a range of proteins that are toxic to insects. We recently identified a novel 42-kDa protein from Xenorhabdus nematophila that was lethal to the larvae of insects such as Galleria mellonella and Helicoverpa armigera when it was injected at doses of 30 to 40 ng/g larvae. In the present work, the toxin gene txp40 was identified in another 59 strains of Xenorhabdus and Photorhabdus, indicating that it is both highly conserved and widespread among these bacteria. Recombinant toxin protein was shown to be active against a variety of insect species by direct injection into the larvae of the lepidopteran species G. mellonella, H. armigera, and Plodia interpunctella and the dipteran species Lucilia cuprina. The protein exhibited significant cytotoxicity against two dipteran cell lines and two lepidopteran cell lines but not against a mammalian cell line. Histological data from H. armigera larvae into which the toxin was injected suggested that the primary site of action of the toxin is the midgut, although some damage to the fat body was also observed.     

荧光发光杆菌TT01品系pirA2B2 基因的克隆表达与杀虫活性

【目的】Photorhabdus luminescens TT01基因组中的一对ORF plu4437-plu4436(简称pirA2B2)的预测氨基酸序列与另一对已证明编码产物有口服杀虫活性的ORF plu4093-plu4092(简称pirA1B1)有50%和45%的一致性,本文旨在研究pirA2B2基因座的表达产物是否也有杀虫活性。【方法】PCR扩增并克隆了pirA2,pirB2和pirA2B2基因,构建了重组表达载体pQE-pirA2,pQE-pirB2和pQE-pirA2B2并分别转入M15菌株表达,经SDS-PAGE和Western blot检测证明,3个重组菌株经IPTG诱导后,分别成功表达了可溶的PirA2,PirB2和PirA2B2蛋白。用亲和层析结合脱盐技术对3个重组菌株表达的外源蛋白分别进行纯化,并通过生物测定确定纯化蛋白的杀虫活性。【结果】生物测定结果显示联合表达的PirA2B2对大蜡螟和斜纹夜蛾五龄幼虫均有明显的血腔杀虫活性,LD50分别为每虫4.0和2.8μg,单独表达的PirA2或PirB2对上述2种害虫没有血腔杀虫活性,但两者的混合物具有与两者联合表达相似的杀虫活性;PirA2B2对大蜡螟和斜纹夜蛾初孵幼虫均无口服杀虫活性。【结论】pirA2B2是P.luminescens TT01菌株基因组中的另一个二元杀虫毒素基因。【意义】pirA2B2的成功克隆表达和杀虫功能的确定为进一步研究其与pirA1B1的关系以及该基因的表达调控等打下了基础。     

对二点委夜蛾高毒力苏云金芽胞杆菌的筛选及其基因型分析

【目的】获得对二点委夜蛾Athetis lepigone(M(o|¨)schler)高毒力的苏云金芽胞杆菌(Bt)菌株,寻找对该虫具有特异杀虫活性的蛋白毒素,探索Bt菌株或其杀虫基因应用于二点委夜蛾防治的可行性。【方法】通过生物测定方法比较了36株苏云金芽胞杆菌和一株恶臭假单胞工程菌PHB-cry1Ab对二点委夜蛾幼虫的杀虫活性,同时利用PCR-RFLP方法对这些菌株的基因型进行了分析。【结果】不同Bt菌株对二点委夜蛾幼虫的杀虫活性差别很大,杀虫活性高的菌株都含有cry1Ac基因。饲毒72 h后含单基因的BtHD-73菌株(cry1Ac)对二点委夜蛾2龄幼虫的毒力(LC_(50)值为188.51μg/g)明显高于含多基因的Bt SC-40菌株(cry1Ac,cry2Ac,cry1I,vip3A)的毒力(LC_(50)值为418.13μg/g)。含有vip3A基因的Bt SC-40和BtHD-13营养期上清液对二点委夜蛾2龄幼虫表现出一定的杀虫活性(72 h死亡率分别达到42.5%和57.4%),而无vip3A基因的Bt HD-73营养期上清液未表现出明显的杀虫活性。【结论】由cry1Ac基因编码的Cry1Ac蛋白对二点委夜蛾幼虫具有特异杀虫活性,Vip3A蛋白对二点委夜蛾幼虫可能也有一定的杀虫活性。     

Molecular analysis of a novel gene cluster encoding an insect toxin in plant-associated strains of Pseudomonas fluorescens

Pseudomonas fluorescens CHA0 and the related strain Pf-5 are well-characterized representatives of rhizosphere bacteria that have the capacity to protect crop plants from fungal root diseases, mainly by releasing a variety of exoproducts that are toxic to plant pathogenic fungi. Here, we report that the two plant-beneficial pseudomonads also exhibit potent insecticidal activity. Anti-insect activity is linked to a novel genomic locus encoding a large protein toxin termed Fit (for P.   f luorescens i nsecticidal t oxin) that is related to the insect toxin Mcf ( M akes c aterpillars f loppy) of the entomopathogen Photorhabdus luminescens , a mutualist of insect-invading nematodes. When injected into the haemocoel, even low doses of P. fluorescens CHA0 or Pf-5 killed larvae of the tobacco hornworm Manduca sexta and the greater wax moth Galleria mellonella . In contrast, mutants of CHA0 or Pf-5 with deletions in the Fit toxin gene were significantly less virulent to the larvae. When expressed from an inducible promoter in a non-toxic Escherichia coli host, the Fit toxin gene was sufficient to render the bacterium toxic to both insect hosts. Our findings establish the Fit gene products of P. fluorescens CHA0 and Pf-5 as potent insect toxins that define previously unappreciated anti-insect properties of these plant-colonizing bacteria.     

塑料饲养大蜡螟幼虫肠道可培养细菌多样性

【背景】昆虫肠道中存在大量的微生物,是昆虫正常生命活动所必需的。它们能够促进维生素的合成、脂肪和碳水化合物的吸收及利用,同时还可以保护宿主抵御天敌,忍受高温以及促进毒素或异生素的代谢,间接促进资源开发。【目的】研究PE塑料饲喂的大蜡螟幼虫肠道可培养细菌的多样性。【方法】利用16S rRNA基因序列分析技术,结合菌落形态和细胞形态及相关生理生化特征鉴定细菌种类。【结果】从大蜡螟幼虫肠道分离纯化的40株可培养细菌得到16种不同细菌遗传型,分别属于芽孢杆菌科(Bacillaceae)、肠球菌科(Enterococcaceae)、葡萄球菌科(Staphylococcaceae)、莫拉菌科(Moraxellaceae)4个科。其中芽孢杆菌科是肠道可培养细菌的优势细菌种类。结合菌落和细胞形态及生理生化特征,确定肠道可培养细菌为芽孢杆菌属9株、肠球菌属4株、葡萄球菌属2株以及不动杆菌属1株。【结论】通过研究大蜡螟幼虫肠道可培养细菌群落结构组成,可为开展大蜡螟肠道的微生态研究提供相关理论基础。     

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.