苏云金芽孢杆菌杀线虫晶体蛋白的相关基因

本文从苏云金杆菌对线虫活性的发现、编码毒蛋白的基因、毒蛋白的种类大小及苏云金杆菌对线虫的作用机理4个方面进行了综述，并简要展望了其研究和应用前景。     

具杀松材线虫活性的苏云金芽胞杆菌筛选及其毒力测定

松材线虫是松树萎蔫病的主要病原,为了筛选到具有较高杀线虫活性的苏云金芽胞杆菌Bacillus thuringiensis菌株,采用直接触杀法对苏云金芽胞杆菌发酵液进行杀线虫活性以及稳定性的检测,并对筛选出的高活性菌株通过扩增16S rDNA片段进行菌株的鉴定。结果表明,4个菌株中具有最高活性的菌株D发酵液处理松材线虫8 h的死亡率超过70%,24 h其死亡率达到100%;在温度为-20、25、40、60、80和121℃条件下处理的菌株D发酵液,其对松材线虫的毒力都在85%以上;在pH 5.0~10.0时,菌株D发酵液对松材线虫的毒力都在85%~94%,并无显著的差异;将菌株D发酵液进行蛋白酶K处理后,其对松材线虫的毒力仍在98%以上;在4℃环境下保存50 d,菌株D发酵液的毒力没有明显下降。通过16S rDNA 1.5 kb片段的序列分析,初步得出菌株D可能是苏云金芽胞杆菌KNU-07。可见,菌株D发酵液具有耐热、耐储藏和对蛋白酶K稳定的特性,是一株具有开发潜力和应用前景的高毒力菌株。     

苏云金芽孢杆菌防治鞘翅目害虫的研究进展

本文综述了国内外有关防治鞘翅目害虫的苏云金芽孢杆菌菌株、杀虫晶体蛋白及作用方式、生物测定以及制剂和应用等方面的研究进展。     

对植物寄生线虫具有活性的苏云金芽胞杆菌研究进展

植物寄生线虫是重要的植物病原物之一,给农业生产带来了巨大的经济损失。长期以来,一直缺乏有效防治植物寄生线虫的手段。苏云金芽胞杆菌（Bacillus thuringiensis,Bt）是重要的昆虫病原细菌,广泛应用于鳞翅目、双翅目、鞘翅目害虫等农林及卫生害虫的防治;部分Bt菌株对植物寄生线虫具有很高的活性。本文总结了杀植物寄生线虫Bt菌株筛选的模型与方法的建立、Bt菌株杀植物寄生线虫的作用机理及其相关应用、以及杀植物寄生线虫的伴胞晶体蛋白与相关基因等,可望为杀植物寄生线虫Bt制剂的研究与开发提供借鉴。     

苏云金芽孢杆菌晶体蛋白对北方根结线虫生物测定方法的建立和杀虫效果评价

 A bioassay procedure was developed to assess the toxicity of Bacillus thuringiensis crystal protein against Meloidogyne hapla,a root-knot nematode,under laboratory conditions.Reproducibility and precision of the bioassay results were optimal when forty 2nd stage juveniles were incubated in the dissolved crystal protein solution at 25℃,pH9.0 for 7 days.The juveniles were stained with 1% KMnO₄ for 2 hours or methylene blue solution for 1 hour to distinguish living and dead ones.By the bioassay procedure,the LC₅₀ value of strain YBT-1532 crystal protein against M.hapla was determined as 0.304±0.086 mg/mL(LC₅₀±1.96SE).Moreover,the strain YBT-1532 showed toxicity to Caenorhabditis elegans,a free-living nematode.All results indicated that YBT-1532 is a toxic strain to plant-parasitic nematode,and has the potential to control plant-parasitic nematode.     

重组杀蚊苏云金芽孢杆菌研究进展

苏云金芽孢杆菌除了对鳞翅目多种昆虫具有毒杀作用外,还有多种亚种或血清型对蚊虫也具有杀虫活性。目前,将多种杀蚊毒素基因进行重组表达,取得了一定的效果。本文就近年来有关杀蚊苏云金芽孢杆菌及其晶体蛋白的结构、分子作用机理,以及利用DNA重组技术提高杀蚊效果的研究进展作一简述。     

具杀线虫活性的苏云金杆菌筛选研究

 Bioassay of 51 strains of Bacillus thuringiensis to Bursaphelenchus xylophilus had been studied in laboratory. Two of them had higher toxicity to B. xylophilus. Extraction of endotoxin and exotoxin of the two isolates to B. xylophilus showed the action of exotoxin higher than that of endotoxin.     

对夜蛾科幼虫高毒力的苏云金芽孢杆菌菌株及其晶体蛋白特性

本文对自德国土壤中分离的８株苏云金芽孢杆菌进行毒力测定和晶体蛋白特性的研究。生物测定结果表明，８株Ｂｔ中除Ｓ２１－１、Ｓ２１－１３对甜菜夜蛾无毒性外，对夜蛾科的４种供试昆虫均有很高的毒效，４８小时校正死亡率高达８０％～１００％；扫描电镜观察和ＳＤＳ－ＰＡＧＥ晶体蛋白电泳结果可知，Ｓ１１－６和Ｓ２１－１３２个菌株只有一种菱形晶体和一条相应的１３５ＫＤａ主带，其余６个菌株均有菱形和方形两种晶体，以及两条相应的１３５ＫＤａ和６５ＫＤａ主带。另对晶体的特性与其毒力的关系进行了讨论。     

三种苏云金芽孢杆菌晶体蛋白火箭免疫电泳的研究

本文以火箭免疫电泳技术检测HD—1、81—6、187三种苏云金芽孢杆菌的晶体蛋白,结果表明:这种方法可以快速、特异性地检测晶体蛋白的含量。在抗血清浓度为3％的凝胶上抗原抗体所形成的免疫沉淀线峰高与晶体蛋白的浓度呈明显的线性关系。本文还就火箭免疫电泳沉淀线峰高与一些条件的关系进行了讨论。     

苏云金芽孢杆菌培养条件及晶体蛋白提纯方法初探

在苏云金芽孢杆菌常用培养基的基础上,设计了３种改进苏云金芽孢杆菌产生伴孢晶体的培养基,从中筛选出一种比１／２ＬＢ、Ｇ Ｔ培养基培养周期短（约需３８ｈ）的ＺＭ培养基。同时,改进了晶体蛋白的提纯方法—碱裂解法。改进后的方法与不连续蔗糖密度梯度离心法相比,简便易行,提取量大,在Ｂｔ的生化和生物活性研究上具有一定参考价值。     

Induction of resistance against pine wilt disease caused by Bursaphelenchus xylophilus using selected pine endophytic bacteria

Pine wilt disease (PWD) is the most destructive disease threatening pine worldwide. The disease is mainly caused by the pine wood nematode, Bursaphelenchus xylophilus, which is vectored by pine sawyer longhorn-beetles, Monochamus spp. This study aimed to select resistance-inducing pine endophytic bacteria for management of PWD. To set up a defence-related genes expression pattern for screening, four chemical inducers (salicylic acid, γ-aminobutyric acid (GABA), β-aminobutyric acid and α-aminobutyric acid) were tested in vitro on pine calli and in vivo on pine seedlings. Treatment with GABA had the greatest reduction in PWD severity on pine seedlings. The pattern of defence-related gene expression in calli treated with GABA was used to select potential resistance-inducing bacterial strains. In addition, 92 bacterial strains were isolated from pine tree needles and stems and were tested for expression of defence-related genes in pine calli in vitro. Among the tested strains, 13 showed a similar pattern to GABA treatment in at least four tested defence-related genes and were selected for the seedling assay. From the seedling assay, three bacterial strains (16YSM-E48, 16YSM-P180 and 16YSM-P39) showed significant reduction in PWD severity compared to the untreated control. Moreover, among the selected strains, cell-free culture supernatant of strain 16YSM-P180 significantly reduced PWD severity in inoculated pine seedlings. The selected strains were identified based on the 16S rRNA sequence as Pseudomonas putida 16YSM-E48, Curtobacterium pusillum 16YSM-P180 and Stenotrophomonas rhizophila 16YSM-P39. These selected strains are suggested as potential alternatives for management of PWD by induction of systemic resistance.     

松材线虫伴生细菌与松树萎蔫病关系的初步研究

利用马尾松水培离体松枝作接种材料,接种消毒后的松材线虫、线虫虫体上分离到细菌分离物GD1以及两者的混合体。结果表明,单独接种消毒后的松材线虫蔌细菌分离物GD1均不能使松枝萎蔫,只有两者混合接种才能使松枝发病枯死。从而说明了松材线虫的伴生细菌对松树萎蔫病的发生起着重要的作用。     

Emamectin benzoate as a candidate for a trunk‐injection agent against the pine wood nematode,Bursaphelenchus xylophilus

In order to develop an effective trunk‐injection agent against pine wood nematode, Bursaphelenchus xylophilus, an in vitro assay was used to examine the antinematodal activity of 58 commercially available compounds with known modes of action. Among compounds tested, the GABA receptor agonists had better anti‐nematodal activity than compounds influencing glutamate, N‐methyl‐D ‐aspartate, β‐adrenergic, dopamine, muscarinic acetylcholine and nicotinic acetylcholine receptors, as well as those inhibiting acetylcholinesterase, monoamine oxidase, 5‐hydroxytryptamine uptake and Ca²⁺, K⁺, Na⁺ and Cl⁻ channels. Avermectins and milbemycins strongly inhibited propagation of the nematode. Emamectin benzoate proved to be the most active (IC₉₅ 0.050 µM ) being over 140 times more active than the active ingredient of conventional trunk‐injection agents. It is concluded that emamectin benzoate is a strong candidate for an anti‐nematodal trunk injection agent. © 2000 Society of Chemical Industry     

Duplex PCR 快速检测松材线虫

利用duplex PCR技术对松材线虫(Bursaphelenchus xylophilus)与拟松材线虫(B. mucronatus)的rDNA部分核苷酸序列扩增.根据松材线虫与拟松材线虫的ITS1序列区别,设计出特异性引物,检测松材线虫的存在;在5.8S,28S保守序列区设计通用引物,检测伞滑刃线虫的存在.     

Development of a water-soluble preparation of emamectin benzoate and its preventative effect against the wilting of pot-grown pine trees inoculated with the pine wood nematode, Bursaphelenchus xylophilus

Water-soluble preparations have been investigated to develop a trunk injection agent based on the poorly water-soluble anti-nematode emamectin benzoate. Following tests on the phytotoxicity of some solvents and solubilizers and demonstration of the ability of some solubilizers to dissolve emamectin benzoate in water, acetone + methanol was selected as the solvent and Polysorbate 80 as the solubilizer. This water-soluble preparation of emamectin benzoate prevented the wilting of pot-grown 4-year-old trees of the Japanese black pine, Pinus thunbergii, artificially inoculated with the pine wood nematode, Bursaphelenchus xylophilus, at a dose of 20 g emamectin benzoate per cubic metre of pine tree.     

松材线虫类毒过敏原蛋白的原核表达及多克隆抗体的制备

 利用重叠延伸PCR基因合成法,按大肠杆菌偏爱密码子,合成松材线虫类毒过敏原蛋白基因（Bxvap2）,将其连接到原核表达载体pET-29b(+)上。获得的重组子转化大肠杆菌BL21(DE3)后,用IPTG进行诱导表达。SDS-PAGE分析表明,目的蛋白以可溶形式高效表达,占细菌总蛋白的33.5 %。将表达产物经Ni-NTA亲和柱纯化后进行质谱分析,结果显示纯化的蛋白为BXVAP2蛋白。用制备的BXVAP2蛋白免疫新西兰白兔制备多克隆抗体,间接ELISA测得抗体灵敏度5.4 ng·mL^-1（效价为1∶1 360 000）,为进一步研究该蛋白的组织定位及功能,明确该基因在致病过程中的作用机理奠定了基础。