昆虫病原斯氏线虫SY-5对重组Cip晶体蛋白的营养利用（英文稿）

【目的】初生型Photorhabdus luminescens细菌产生两种胞内晶体蛋白CipA和CipB,为其共生的昆虫病原异小杆线虫提供营养。探索非共生的斯氏线虫对Cip蛋白的营养利用情况。【方法】在已构建重组Cip蛋白大肠杆菌表达体系的基础上,建立重组菌细胞与无菌斯氏SY-5线虫共培养系统,检测线虫的生长发育情况。【结果】Cip蛋白对目标线虫生长有显著支持作用:发育为成虫的比例达到65%-82%,雌虫的怀卵率为80%-95%,平均怀卵量为30-50粒,并显著降低各虫态的死亡率。【结论】Cip蛋白不仅为共生的异小杆线虫提供营养,亦能为斯氏线虫所利用。     

重组昆虫病原细菌Cip蛋白基因工程菌发酵条件的初步研究

昆虫病原细菌产生两种胞内晶体蛋白CipA和CipB,为新型的生物农药——昆虫病原线虫提供必需的营养。在已构建原核表达载体pET-15b-cipA和pET-15b-cipB的基础上,研究了这两种重组载体在不同宿主菌中的表达情况,重组菌的生长特性,摇瓶培养时表达重组Cip蛋白工程菌的发酵条件。结果显示:以E.coli BL21(DE3)为宿主菌,在LB培养基中培养至OD600为0.8～1.0时,1 mmol/L IPTG诱导8 h,CipA和CipB的表达量可达30.9%和32.6%,重组质粒具有良好的分离稳定性和结构稳定性。     

昆虫病原线虫的共生细菌

昆虫病原线虫与其共生细菌二者互惠共生 :共生细菌需要昆虫病原线虫作为载体以寄生寄主昆虫并做为自己的营养来源 ,而昆虫病原线则需要依靠共生细菌来杀死昆虫。综述了共生细菌的病原作用、抗菌作用与杀虫作用 ,评述了共生细菌的基因工程进展 ,讨论了昆虫共生细菌在昆虫病原线虫致病性的作用。     

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。     

昆虫病原线虫与其共生细菌共生关系的研究进展

昆虫病原斯氏和异小杆线虫与共生细菌的共生关系是这类线虫作为害虫生物防治因子的基础。从线虫共生细菌的信息、营养、抗菌和病原作用,以及线虫对共生细菌的保护和媒介作用综述昆虫病原线虫与其共生细菌的共生关系;描述这一共生关系的影响因子;同时,讨论了未来的研究方向和应用前景。     

基于启动子和宿主改造的酿酒酵母表达系统优化研究

生物医药领域中一套高效表达系统对于重组蛋白的生产至关重要。酿酒酵母作为一种食品级真核微生物,具有繁殖迅速、培养简单、遗传操作便捷等特点,是生产重组蛋白较理想的表达系统之一。对实验室已有的p HR酿酒酵母表达系统进行优化。分别通过易错PCR技术和菌株诱变技术对酿酒酵母启动子PTEF和宿主酿酒酵母Y16进行突变改造,经筛选、鉴定获得表达性能提高的启动子PTEFV1和酿酒酵母Y16-E14、Y16-E19。随后,利用启动子PTEFV1构建以Y16-E14为宿主的p HR-N酿酒酵母表达系统,以绿色荧光蛋白和人血清白蛋白为对象,比较表达系统改造前后性能变化。结果显示p HR-N酿酒酵母表达系统无论胞内表达绿色荧光蛋白还是分泌表达人血清白蛋白的能力均较改造前明显提高。p HR-N系统为获得更多具有重要应用价值的重组蛋白提供了有利的工具。     

HBV preS/S基因克隆及其在酿酒酵母中的表达

目的:探索利用酿酒酵母系统表达乙型肝炎病毒(HBV)preS／S基因。方法:利用PCR技 术,以HBV病毒DNA为模板,体外扩增HBV preS／S基因。然后构建重组表达载体pESC-preS／S。 用LiAc法转化酿酒酵母YPH50,选取重组菌进行培养,并诱导表达外源蛋白。提取蛋白浓缩后 进行SDS-PAGE分析,并经Western blot分析鉴定。结果:实验结果表明重组菌能够表达HBV preS／S蛋白。结论:利用酿酒酵母系统可成功表达HBV preS／S基因,为制备新型预防性疫苗提供 条件。     

木糖异构酶在酿酒酵母细胞表面的展示

将来源于嗜热细菌Thermus thermophilus的木糖异构酶基因xylA,与酿酒酵母(Sac-charomyces cerevisiae)a-凝集素表面展示载体pYD1的Aga2p亚基C端序列融合。编码融合蛋白的基因序列前接上半乳糖诱导型启动子。用LiAc完整细胞法转化酿酒酵母EBY100。含重组质粒的菌株EBY100/pYD-xylA经半乳糖诱导表达外源融合蛋白,免疫荧光显微镜结果显示外源蛋白被锚定在细胞壁上,木糖异构酶活性测定结果表明,细胞壁上酶活测定值为1.52U,木糖异构酶在酿酒酵母细胞壁上得到活性表达。     

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。     

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…     

Nutritive significance of crystalline inclusion proteins of Photorhabdus luminescens in Steinernema nematodes

Phase I cells of Photorhabdus luminescens produce two types of intracellular crystalline inclusion proteins designated CipA and CipB. The genes encoding CipA and CipB proteins from P. luminescens H06 were expressed respectively in Escherichia coli and these cells were used to feed the axenic first juveniles (J1) of three Steinernema nematode isolates in liquid cultures and on agar plates. In liquid cultures, the axenic J1 juveniles of all three test Steinernema nematode isolates were able to produce next dauer juveniles (DJs) in the E. coli cultures with at least one of the expressed Cip proteins, but unable to develop beyond the next J1 stage without expressed Cip proteins. For each target nematode isolate, addition of the supernatant of the bacterial culture of its Xenorhabdus symbiont to the tested liquid cultures did not induce the formation of DJs. However, on LB agar plates with different test E. coli cultures, all J1 juveniles of the three Steinernema strains finally developed into next DJs. It seemed that the metabolite pathway of the test bacteria in both culture systems was different. The presence of the Cip proteins has a significant influence on the DJ formation of the Steinernema nematodes in liquid culture system.     

Coprinus comatus damages nematode cuticles mechanically with spiny balls and produces potent toxins to immobilize nematodes

We reported recently a unique fungal structure, called the spiny ball, on the vegetative hyphae of Coprinus comatus (O. F. Müll.:Fr.) Pers. Although some observations regarding the role of this structure were presented, its function remained largely unknown. In this study, we showed that purified (isolated and washed) spiny balls could immobilize and kill the free-living nematode Panagrellus redivivus Goodey highly efficiently. Scanning electron microscopy studies illustrated that the spiny structure damaged the nematode cuticle, suggesting the presence of a mechanical force during the process of nematode immobilization. Severe injuries on nematode cuticles caused the leakage of inner materials of the nematodes. When these structures were ground in liquid nitrogen, their killing efficacy against nematodes was lost, indicating that the shape and the complete structure of the spiny balls are indispensable for their function. However, extraction with organic solvents never lowered their activity against P. redivivus, and the extracts showed no obvious effect on the nematode. We also investigated whether C. comatus was able to produce toxins which would aid in the immobilization of nematodes. In total, we identified seven toxins from C. comatus that showed activity to immobilize the nematodes P. redivivus and Meloidogyne incognita (Kofoid et White) Chitwood. The chemical structures of these toxins were identified with nuclear magnetic resonance, mass spectrometry, infrared, and UV spectrum analysis. Two compounds were found to be novel. The toxins found in C. comatus are O-containing heterocyclic compounds.     

Acanthocytes of Stropharia rugosoannulata function as a nematode-attacking device

Efficient killing of nematodes by Stropharia rugosoannulata Farlow ex Murrill cultures was observed. This fungus showed the ability to immobilize the free-living nematode Panagrellus redivivus Goodey within minutes and to immobilize the pine wilt nematode Bursaphelenchus xylophilus (Steiner & Buhrer) Nickle within hours on agar plates. Moreover, P. redivivus worms were completely degraded by the fungus within 24 to 48 h. The cultures of S. rugosoannulata studied shared the characteristic of abundantly producing cells with finger-like projections called acanthocytes. We showed that the nematode-attacking activity of this fungus is carried out by these spiny acanthocytes and that mechanical force is an important factor in the process. Furthermore, the growth and nematode-attacking activity of the fungus in soil were also determined, and our results suggest that acanthocytes are functional in soil.     

Coprinus comatus: A basidiomycete fungus forms novel spiny structures and infects nematode

Nematophagous basidiomycete fungi kill nematodes by trapping, endoparasitizing and producing toxin. In our studies Coprinus comatus (O.F.Müll. : Fr.) Pers. is found to be a nematode-destroying fungus; this fungus immobilizes, kills and uses free-living nematode Panagrellus redivivus Goodey and root-knot nematode Meloidogyne arenaria Neal. C. comatus produces an unusual structure designated spiny ball. Set on a sporophore-like branch, the spiny ball is a burr-like structure assembled with a large number of tiny tubes. Purified spiny balls exhibit moderate nematicidal activity. Experiments show that spiny balls are not chlamydospores because of the absence of nuclei in the structures and quick formation within 3 d in a young colony. Nematodes added to C. comatus cultures on potato-dextrose agar (PDA) and cornmeal agar (CMA) become inactive in hours. Infection of nematodes by the fungus occurs only after the nematodes are immobilized (feeble or dead), probably by a toxin. Electron micrographs illustrate that C. comatus infect P. redivivus by producing penetration pegs with which hyphae colonize nematode bodies. An infected nematode is digested and consumed within days and hyphae grow out of the nematode.     

Panagrellus redivivus: failure to find evidence for the occurrence and biosynthesis of sialic acids.

The occurrence of sialic acids in the free-living nematode Panagrellus redivivus was studied by periodate oxidation/[3H]sodium borohydride reduction of about 10(7) nematodes. In parallel, the capability of sialic acid biosynthesis was examined by metabolic labeling of the same number of nematodes with N-[3H]acetylmannosamine. In both experiments, radioactivity was incorporated into the nematodes. Mild acid hydrolysis, however, did not release radioactively labeled sialic acids or derivatives as tested by radio thin-layer chromatography, suggesting that P. redivivus does not contain or synthesize sialic acids.     

Development of a low-cost technology for mass production of the free-living nematode <Emphasis Type="Italic">Panagrellus redivivus</Emphasis> as an alternative live food for first feeding fish larvae

The free-living nematode Panagrellus redivivus is a suitable food source for first feeding fish. In the present report, a new method for the mass production of P. redivivus is presented. The technique involves multiplication of the nematode in monoxenic (single microorganism: Saccharomyces cerevisiae) solid culture (fluid media supported by 1- to 4-cm(3) sponge cubes) in autoclavable plastic bags (size range: 50 x 30 cm to 75 x 67 cm). Two growing media were tested: oat-meal medium (OM), which is an oat-based medium (16.7% oat-meal flour in 0.8% saline solution), and purified ingredient medium (PIM), a semi-synthetic medium (1.64% meat peptone, 0.94% yeast extract, 12.6% corn starch, 0.24% glucose, 1.48% sunflower oil, in 0.8% saline solution). The bags were inoculated with 350 nematodes/g medium. After an average period of 12 days (11-13 days) at 25 degrees C, the average yield (number of nematodes/g medium) was 241 x 10(3) for OM and 333 x 10(3) for PIM in 12-l bags (50 x 30 cm). The production scale has currently reached a bag volume of 50 l (75 x 67 cm); using PIM and the conditions described above, it was possible to harvest more than 1.3 x 10(9) nematodes/bag (291 x 10(3) nematodes/g medium). In PIM, when sun flower oil was replaced with the same amount of fish oil or cod liver oil, yields of 259 x 10(3) and 290 x 10(3) nematodes/g medium, respectively, were attained. The technology for mass production and formulation of P. redivivus should enable fish-hatchery operators to rely on a cheap, standardised, and permanently available live food product for first feeding fish larvae.     

Catecholaminergic structures in the nervous system of three nematode species, with observations on related enzymes

The formaldehyde fluorescence technique has been used to demonstrate amine-specific fluorescence in the nervous system of three nematode species, Prionchulus punclatus Cobb, Panagrellus redivivus Goodey, and Aphelenchus avenae Bastian. Examination of some of the physical and chemical properties of this fluorescence has shown it to be due principally to the primary catecholamine dopamine. Dopamine and dopa decarboxylase were also detected biochemically in A. avenae. Dopamine has now been proposed as a putative neurotransmitter in a number of nematode species and the role of this and other biogenic amines in nematodes is discussed. Of the two principal enzymes involved in the metabolism of monoamines, catechol-O-methyltransferase was detected in both A. avenae and P. redivivus but monoamine oxidase could not be detected in these or other nematode species.