Heterorhabditis bacteriophora and Steinernema carpocapsae are microscopic entomoparasitic nematodes (EPNs) that are attractive, organic alternatives for controlling a wide range of crop insect pests. EPNs evolved with...Heterorhabditis bacteriophora and Steinernema carpocapsae are microscopic entomoparasitic nematodes (EPNs) that are attractive, organic alternatives for controlling a wide range of crop insect pests. EPNs evolved with parasitic adaptations that enable them to “feast” upon insect hosts. The infective juvenile, a non-feeding, developmentally arrested nematode stage, is destined to seek out insect hosts and initiates parasitism. After an insect host is located, EPNs enter the insect body through natural openings or by cuticle penetration. Upon access to the insect hemolymph, bacterial symbionts (Photorhabdus luminescens for H. bacteriophora and Xenorhabdus nematophila for S. carpocapsae) are regurgitated from the nematode gut and rapidly proliferate. During population growth, bacterial symbionts secrete numerous toxins and degradative enzymes that exterminate and bioconvert the host insect. During development and reproduction, EPNs obtain their nutrition by feeding upon both the bioconverted host and proliferated symbiont. Throughout the EPN life cycle, similar characteristics are seen. In general, EPNs are analogous to each other by the fact that their life cycle consists of five stages of development. Furthermore, reproduction is much more complex and varies between genera and species. In other words, infective juveniles of S. carpocapsae are destined to become males and females, whereas H. bacteriophora develop into hermaphrodites that produce subsequent generations of males and females. Other differences include insect host range, population growth rates, specificity of bacterial phase variants, etc. This review attempts to compare EPNs, their bacterial counterparts and symbiotic relationships for further enhancement of mass producing EPNs in liquid media.展开更多
【背景】光杆菌存在于嗜菌异小杆线虫肠道内,并与其互惠共生,其能够产生多种高效、广谱的杀虫蛋白及毒素,是近年来继苏云金芽胞杆菌(Bt)之后挖掘新型杀虫蛋白及杀虫基因的热点研究对象。【目的】克隆Photorhabdus luminescens(NLK-1)Tx...【背景】光杆菌存在于嗜菌异小杆线虫肠道内,并与其互惠共生,其能够产生多种高效、广谱的杀虫蛋白及毒素,是近年来继苏云金芽胞杆菌(Bt)之后挖掘新型杀虫蛋白及杀虫基因的热点研究对象。【目的】克隆Photorhabdus luminescens(NLK-1)Txp40毒蛋白基因,分析其与已知其他同属共生菌相似毒蛋白在基因序列、蛋白组成、理化性质及构象的区别,构建原核表达载体并转化大肠杆菌进行诱导表达,初步测定其杀虫活性。【方法】采用侵染的大蜡螟幼虫血腔直接分离初生型共生细菌,根据已报道的序列经比对分析设计引物,扩增目的基因,连接克隆质粒p MD19-T后测序,利用Expasy在线Prot Param tool预测其基本理化特性参数,NPS@-Network Protein Sequence Analysis在线工具进行二级结构预测。通过克隆、酶切、连接目的基因在p ET28a原核表达载体上,转化大肠杆菌BL21中,利用蓝白斑筛选阳性克隆,测序验证后进行IPTG诱导表达;菌体超声破碎离心,以毒蛋白含量较高的上清溶液对大蜡螟幼虫进行饲喂和血腔注射毒性测定。【结果】Photorhabdus luminescens(NLK-1)Txp40毒蛋白基因全长为1 008 bp,与已知相关基因的序列相似性为94%,与已知40 k D相关蛋白的氨基酸相似性达到99%,分子量37.9 k D,p I 8.37,二级结构预测表明其主要由α螺旋35.71%,无规卷曲54.46%,延伸链9.52%组成,跨膜区域与已知蛋白基本相似,克隆构建了原核表达载体p ET28a-(NLK-1)Txp40,SDS-PAGE分析其在38 k D处有特异条带,蛋白分子量与预测值基本一致,且表达相对单一,表达量较高。Photorhabdus luminescens(NLK-1)Txp40蛋白对大蜡螟幼虫具有较高的血腔毒性,大蜡螟幼虫注射5μL蛋白粗提液剂量下48 h内致死率达100%,未发现胃毒活性。【结论】获得Photorhabdus luminescens(NLK-1)Txp40毒蛋白基因,比对、分析了与已知基因在序列组成、蛋白基本理化性质和二级结构的异同,构建�展开更多
文摘Heterorhabditis bacteriophora and Steinernema carpocapsae are microscopic entomoparasitic nematodes (EPNs) that are attractive, organic alternatives for controlling a wide range of crop insect pests. EPNs evolved with parasitic adaptations that enable them to “feast” upon insect hosts. The infective juvenile, a non-feeding, developmentally arrested nematode stage, is destined to seek out insect hosts and initiates parasitism. After an insect host is located, EPNs enter the insect body through natural openings or by cuticle penetration. Upon access to the insect hemolymph, bacterial symbionts (Photorhabdus luminescens for H. bacteriophora and Xenorhabdus nematophila for S. carpocapsae) are regurgitated from the nematode gut and rapidly proliferate. During population growth, bacterial symbionts secrete numerous toxins and degradative enzymes that exterminate and bioconvert the host insect. During development and reproduction, EPNs obtain their nutrition by feeding upon both the bioconverted host and proliferated symbiont. Throughout the EPN life cycle, similar characteristics are seen. In general, EPNs are analogous to each other by the fact that their life cycle consists of five stages of development. Furthermore, reproduction is much more complex and varies between genera and species. In other words, infective juveniles of S. carpocapsae are destined to become males and females, whereas H. bacteriophora develop into hermaphrodites that produce subsequent generations of males and females. Other differences include insect host range, population growth rates, specificity of bacterial phase variants, etc. This review attempts to compare EPNs, their bacterial counterparts and symbiotic relationships for further enhancement of mass producing EPNs in liquid media.
文摘【背景】光杆菌存在于嗜菌异小杆线虫肠道内,并与其互惠共生,其能够产生多种高效、广谱的杀虫蛋白及毒素,是近年来继苏云金芽胞杆菌(Bt)之后挖掘新型杀虫蛋白及杀虫基因的热点研究对象。【目的】克隆Photorhabdus luminescens(NLK-1)Txp40毒蛋白基因,分析其与已知其他同属共生菌相似毒蛋白在基因序列、蛋白组成、理化性质及构象的区别,构建原核表达载体并转化大肠杆菌进行诱导表达,初步测定其杀虫活性。【方法】采用侵染的大蜡螟幼虫血腔直接分离初生型共生细菌,根据已报道的序列经比对分析设计引物,扩增目的基因,连接克隆质粒p MD19-T后测序,利用Expasy在线Prot Param tool预测其基本理化特性参数,NPS@-Network Protein Sequence Analysis在线工具进行二级结构预测。通过克隆、酶切、连接目的基因在p ET28a原核表达载体上,转化大肠杆菌BL21中,利用蓝白斑筛选阳性克隆,测序验证后进行IPTG诱导表达;菌体超声破碎离心,以毒蛋白含量较高的上清溶液对大蜡螟幼虫进行饲喂和血腔注射毒性测定。【结果】Photorhabdus luminescens(NLK-1)Txp40毒蛋白基因全长为1 008 bp,与已知相关基因的序列相似性为94%,与已知40 k D相关蛋白的氨基酸相似性达到99%,分子量37.9 k D,p I 8.37,二级结构预测表明其主要由α螺旋35.71%,无规卷曲54.46%,延伸链9.52%组成,跨膜区域与已知蛋白基本相似,克隆构建了原核表达载体p ET28a-(NLK-1)Txp40,SDS-PAGE分析其在38 k D处有特异条带,蛋白分子量与预测值基本一致,且表达相对单一,表达量较高。Photorhabdus luminescens(NLK-1)Txp40蛋白对大蜡螟幼虫具有较高的血腔毒性,大蜡螟幼虫注射5μL蛋白粗提液剂量下48 h内致死率达100%,未发现胃毒活性。【结论】获得Photorhabdus luminescens(NLK-1)Txp40毒蛋白基因,比对、分析了与已知基因在序列组成、蛋白基本理化性质和二级结构的异同,构建�
