棕尾别麻蝇幼虫肠道溶纤活性蛋白酶的分离纯化和性质

从以富含纤维蛋白的血凝块为食物的棕尾别麻蝇幼虫肠道浸提液中分离纯化出3种具有溶纤活性的蛋白酶,分别命名为BPGFP1,BPGFP2和BPGFP3。其中,BPGFP1由两个分子量分别为32000和30000的亚基组成。BPGFP2和BPGFP3均为单体,分子量分别为40000和28000。这三种蛋白酶具有相似的底物特异性和抑制剂特性。三种蛋白酶均能降解溶纤活性蛋白酶的特异底物纤维蛋白,Chromzym,P,Chromzym UK和S－2288。三种酶还能够强烈降解类胰蛋白酶专一底物Bz-Phe-Val Arg NA,cBz Gly-Pro-Arg NA,Bz-Pro－Phe-Arg NA和Bz-Val-Gly-Arg NA.PMSF,STI,LBTI和SBBI能够对三种蛋白酶活怀有极强的抑制作用。三种溶纤活性蛋白酶均在pH9.0-10.0范围内表现出较高活性。     

棕尾别麻蝇金属硫蛋白的分离纯化及性质分析

将棕尾别麻蝇Boettcherisca peregrina幼虫置于含800 μg/g CdCl₂的食物中取食48 h后,可诱导金属硫蛋白(MT)的合成。诱导处理后的幼虫匀浆上清液经Sephadex G-50分子筛柱、UNO^TM Q1阴离子交换柱和Bio-Gel P-6脱盐柱层析,纯化得到2个亚型,即MT-Ⅰ和MT-Ⅱ。MT-Ⅰ和MT-Ⅱ的分子量均为9 kD,每蛋白分子均含7个Cd和20个巯基,且具254 nm的Cd-SH特征吸收肩。两者的氨基酸组成中,以半胱氨酸含量最高,分别为36.6%和31.8%;而芳香族氨基酸和组氨酸含量甚少,约1%～2%。     

棕尾别麻蝇雄性附腺分泌物的生理功能

棕尾别麻蝇Boettcherisca peregrina Robinean-Desvoidy雄性附腺的分泌物中包含许多生物活性分子,将成熟的雄性附腺分泌物注射到未交配的雌虫体内,发现其能影响雌虫生殖活性的很多方面:如降低雌虫的再次交配率;增加卵巢内的含卵量;缩短交配后雌虫的寿命。另外,还证明附腺分泌物中存在有可抑制革兰氏阳性菌的物质的存在。     

免疫亲和层析法纯化棕尾别麻蝇(Sarcophagaperegrina)幼虫血淋巴凝集素

 报道了利用免疫亲和层析法纯化棕尾别麻蝇幼虫血淋巴凝集素的结果．哺乳动物红细胞能够特异地吸附凝集素．用兔红细胞与麻蝇幼虫血淋巴凝集素形成的复合体免疫供血家兔,得到麻蝇幼虫血淋巴凝集素的抗体．再利用抗体制备亲和吸附柱,通过免疫亲和层析一次性纯化了麻蝇幼虫血淋巴凝集素． Ｓ Ｄ Ｓ Ｐ Ａ Ｇ Ｅ结果显示,该凝集素的分子量约为７３ ｋ Ｄ．这一结果,与用对麻蝇幼虫血淋巴凝集素有抑制作用的糖蛋白—胎球蛋白和甲状腺球蛋白为配基,亲和层析纯化的结果完全相同,表明用这种免疫亲和层析法纯化凝集素是可行的．为不清楚专一性识别糖或专一性识别糖不典型,难于用普通亲和层析纯化的凝集素,提供了一种有效的纯化方法．     

幼虫期染镉对棕尾别麻蝇繁殖力及生殖腺发育的影响

通过在饲料中添加重金属镉(Cd)饲喂棕尾别麻蝇Boettcherisca peregrine的幼虫,研究镉对其成虫繁殖能力和生殖腺发育的影响.结果表明,棕尾别麻蝇幼虫在取食含100 μg/g、200 μg/g、400 μg/g和800 μg／g Cd2的人工饲料后,成虫的羽化率和寿命都明显下降;但镉处理对成虫的交配率无...     

镉和铜离子诱导棕尾别麻蝇幼虫脂质过氧化丙二醛动态变化分析

采用饲料含毒法,测定了棕尾别麻蝇Boettcherisca peregrine 1日龄幼虫连续取食含不同浓度Cd^2＋或Cu^2＋的人工饲料,体内丙二醛含量的变化过程。实验结果表明：二种重金属离子能诱导昆虫的脂质过氧化反应,并且存在浓度依赖效应和时间依赖效应。在0—1600μg/g饲料浓度范围内,丙二醛（malondialdehyde,MDA）含量与幼虫取食Cd^2＋和Cu^2＋的浓度呈线性关系,所求回归方程分别为y=0．2789＋0．0002x和y=0．3141＋0．0002x。当处理浓度为800μg/g时,在0-120h时间内,MDA含量与幼虫取食Cd^2＋和Cu^2＋的时间呈线性关系,回归方程分别为y=0．3041＋0．0042x和y=0．3251＋0．0029x。     

一株棕尾别麻蝇胚胎细胞系的建立及其特性分析

双翅目昆虫细胞系广泛应用于遗传学、发育生物学、分子生物学、人和动物体病原学以及昆虫抗微生物肽的研究。本研究建立了一株新的棕尾别麻蝇Sarcophaga peregrina胚胎细胞系。该细胞系的原代培养始于2008年11月17日, 取材于棕尾别麻蝇晚期胚胎组织, 在Shields & Sang M3昆虫培养基中于28℃恒温培养, 在第26天进行第1次传代, 至今已历时21个月, 传代72次, 生长状态稳定, 被命名为Sp-E-HNU11。该细胞系的细胞形态主要呈梭形和近圆形, 杂以少量巨型细胞, 紧密贴壁生长。细胞群体倍增时间为42 h。染色体数目一般为10条或12条, 为二倍体或亚二倍体细胞系; 除一对颗粒状微型染色体外, 其他染色体呈短杆状。细胞系的β-萘酯酶和谷草转氨酶同工酶谱上分别显示出1条和3条酶带。随机引物扩增多态性 (random amplified polymorphic DNA, RAPD) 分析结果显示, 该细胞系与小菜蛾细胞系Px-E-HNU12、草地贪夜蛾细胞系IPLB-Sf-9和家蚕细胞系Bm-21E-HNU5呈现明显不同的带型特征。 Sp-E-HNU11细胞系的建立为昆虫抗微生物肽及其他相关的研究工作增添了新的研究工具和生产载体。     

重金属镉与铜在棕尾别麻蝇体内的积累和排泄研究

为了研究镉(Cadmium,Cd)和铜(Copper,Cu)在棕尾别麻蝇Boettcherisca peregrina (Diptera:Sarcophagidae)生长发育过程中积累和排泄的动态变化,在室内给棕尾别麻蝇初产幼虫连续饲喂含不同浓度Cd2'或Cu2+的人工饲料,每隔24 h测定幼虫对Cd2+或Cu2+积累和排泄及变态过程中的变化.结果表明:棕尾别麻蝇幼虫取食含有Cd2+的饲料后,大部分Cd2+积累在体内,少部分通过排泄系统排出.Cd2+在幼虫体内的积累量随着取食时间的延长而增加,幼虫取食含200 μg/g Cd2+饲料后,体内Cd2+积累量高于取食含100 μg/g、400 μg/g和800 μg/g Cd2+饲料后体内Cd2+积累量,幼虫对Cd2+排泄量亦有相同的规律.Cd2+在幼虫、蛹和刚羽化成虫中的含量依次降低.棕尾别麻蝇幼虫取食含Cu2+的饲料后,其体内Cu2+积累量小于排泄量,而且体内积累量和排泄量随着取食时间的延长而增加,Cu2+积累量和排泄量与取食饲料中的Cu2+浓度均不成正比.在变态过程中,Cu2+也不断被排泄,体内Cu2+含量随之下降.     

取食重金属铜对棕尾别麻蝇亲代及子代生长发育与繁殖的影响

为了评估取食含重金属铜饲料对棕尾别麻蝇Boettcherisca peregrine亲代及子代的生长发育与繁殖的影响,在室内给棕尾别麻蝇初产幼虫饲喂含不同浓度（200, 400,800和1 600 µg/g）Cu²⁺的饲料直至化蛹,并对亲代和子代的生长发育和繁殖有关指标进行了观察和分析。结果表明：低浓度的Cu²⁺（200 µg/g）对其体重和体长起促进作用,但对幼虫历期、化蛹率、蛹历期、羽化率、性比、交配率和产仔量无显著作用;较高浓度的Cu²⁺ 则有抑制作用,且Cu²⁺处理浓度越高,亲代幼虫、蛹和雌雄成虫的体重越轻,幼虫和蛹的体长越短,化蛹率、羽化率、交配率和产仔量越低,幼虫历期和蛹期越长,成虫寿命越短。但Cu²⁺处理对成虫性比则无显著的影响。相比之下,经Cu²⁺处理后雌虫所产的子代若不再经Cu²⁺处理,其子代生存、生长发育与繁殖则基本不受影响,说明Cu²⁺对亲代的影响不能遗传至子代。此外,还探讨了该蝇亲代与子代体内Cu²⁺含量在其变态过程中的变化。     

Isolation and characterisation of Beauveria bassiana isolates from phylloplanes of hedgerow vegetation

A leaf imprinting technique combined with a selective medium was used to document the natural occurrence of Beauveria bassiana on phylloplanes of typical hedgerow plants (grasses, stinging nettle and hawthorn) in May, July and September in a hedgerow in Denmark. The density of B. bassiana (as measured by numbers of colony forming units) was greatest in September and on lower nettle leaves. B. bassiana was isolated from phylloplanes in a different hedgerow the following year and a similar picture of occurrence was found. Genetic diversity of selected in vitro isolates were characterised by Universally Primed (UP) PCR, and 13 distinguishable banding patterns were found at the two localities. Of these, four were shared between the field sites and all plant species harboured isolates of B. bassiana with at least two different banding patterns. The isolation method described represents a valuable tool for studying naturally occurring B. bassiana and for rapid isolation of indigenous strains of the fungus for future development of biocontrol agents. The significance of the findings for the life-cycle of B. bassiana is discussed.     

Increased insect virulence in Beauveria bassiana strains overexpressing an engineered chitinase

Entomopathogenic fungi are currently being used for the control of several insect pests as alternatives or supplements to chemical insecticides. Improvements in virulence and speed of kill can be achieved by understanding the mechanisms of fungal pathogenesis and genetically modifying targeted genes, thus improving the commercial efficacy of these biocontrol agents. Entomopathogenic fungi, such as Beauveria bassiana, penetrate the insect cuticle utilizing a plethora of hydrolytic enzymes, including chitinases, which are important virulence factors. Two chitinases (Bbchit1 and Bbchit2) have previously been characterized in B. bassiana, neither of which possesses chitin-binding domains. Here we report the construction and characterization of several B. bassiana hybrid chitinases where the chitinase Bbchit1 was fused to chitin-binding domains derived from plant, bacterial, or insect sources. A hybrid chitinase containing the chitin-binding domain (BmChBD) from the silkworm Bombyx mori chitinase fused to Bbchit1 showed the greatest ability to bind to chitin compared to other hybrid chitinases. This hybrid chitinase gene (Bbchit1-BmChBD) was then placed under the control of a fungal constitutive promoter (gpd-Bbchit1-BmChBD) and transformed into B. bassiana. Insect bioassays showed a 23% reduction in time to death in the transformant compared to the wild-type fungus. This transformant also showed greater virulence than another construct (gpd-Bbchit1) with the same constitutive promoter but lacking the chitin-binding domain. We utilized a strategy where genetic components of the host insect can be incorporated into the fungal pathogen in order to increase host cuticle penetration ability.     

Germination polarity of Beauveria bassiana conidia and its possible correlation with virulence

Three different germination types of conidia; unidirectional, bidirectional and multidirectional, were revealed through microscopic observations for eight Beauveria bassiana isolates germinated on Sabouraud dextrose agar. Canonical correlation analysis indicated that there is a positive correlation between unidirectional germination and virulence against diamondback moth, Plutella xylostella and the Colorado potato beetle, Leptinotarsa decemlineata. Scanning electron microscopy revealed different in vivo behaviors for unipolar- and bipolar-germinated conidia. Unipolar-germinated conidia produced a strong germ tube with mostly appressorium-like structures while bipolar-germinated conidia continued to invasive hyphal growth without any penetration, indicating that germination polarity in one way or another may be an indicator of pathogenic ability.     

Biosynthesis of the cyclooligomer depsipeptide bassianolide,an insecticidal virulence factor of Beauveria bassiana

Beauveria bassiana is a facultative entomopathogen with an extremely broad host range that is used as a commercial biopesticide for the control of insects of agricultural, veterinary and medical significance. B. bassiana produces bassianolide, a cyclooligomer depsipeptide secondary metabolite. We have cloned the bbBsls gene of B. bassiana encoding a nonribosomal peptide synthetase (NRPS). Targeted inactivation of the B. bassiana genomic copy of bbBsls abolished bassianolide production, but did not affect the biosynthesis of beauvericin, another cyclodepsipeptide produced by the strain. Comparative sequence analysis of the BbBSLS bassianolide synthetase revealed enzymatic domains for the iterative synthesis of an enzyme-bound dipeptidol monomer intermediate from d-2-hydroxyisovalerate and l-leucine. Further BbBSLS domains are predicted to catalyze the formation of the cyclic tetrameric ester bassianolide by recursive condensations of this monomer. Comparative infection assays against three selected insect hosts established bassianolide as a highly significant virulence factor of B. bassiana.     

Roles of autophagy-related genes in conidiogenesis and blastospore formation,virulence, and stress response of Beauveria bassiana

Autophagy is a conserved intracellular recycling mechanism, in which autophagy-related genes 12 and 16 (ATG12 and ATG16) function in a complex controlling the ubiquitin-like conjugation system. In the insect-pathogenic fungus Beauveria bassiana, ATG12 and ATG16 were functionally characterized. Disruption of BbATG12 or BbATG16 resulted in the absence of autophagic bodies under starvation stress. ΔBbATG12 and ΔBbATG16 mutant strains displayed similar defects in asexual development (conidiation and blastospore formation) and tolerance to oxidative stress. ΔBbATG16 strain exhibited the impaired growth on the media with gelatin or chitin as a single nitrogen source, and ΔBbATG12 displayed decreased growth on the media with sucrose, fructose or maltose as a single carbon source. Both BbATG12 and BbATG16 were required for fungal virulence. BbATG16 mutation had more effects on fungal virulence than BbATG12 in topical infection assay, although both genes had similar contributions to fungal virulence in intrahemocoel injection assay. This study indicates that BbATG12 and BbATG16 mediate diverse biological functions in addition to their convergent roles in autophagy.     

代谢组学方法鉴定球孢白僵菌孢子萌发和杀虫毒力相关的标记物

【目的】识别球孢白僵菌萌发和杀虫毒力相关的标记物。【方法】本研究对7个球孢白僵菌菌株的孢子进行萌发率和对茶毛虫毒力进行测定,采用基于LC-MS的代谢组学方法,识别菌丝和孢子提取物中与萌发和毒力有关的标记物。【结果】高毒力菌株的菌丝中具有高含量的carnitine、hercynine、acetylcarnitine、α,α-trehalose;Octa-Me、arg-arg-gln、phosphatidylethanolamine[PE(18∶2/0∶0)]、phosphotidylcholine[PC(18∶3/0∶0)]和PC(18∶2/0∶0)。高萌发率菌株的孢子中具有高含量的2,3-dimethylmaleate、acetylcarnitine、propionyl-carnitine和PC(18∶2/0∶0)。高毒力菌株的孢子中具有高含量的histamine、2,5-pyrrolidinedicarboxylic acid;Diamide、carnitine、acetylcarnitine、propionyl-carnitine、butyrylcarnitine、PE(18∶2/0∶0)、PC(16∶1/0∶0)和PC(18∶3/0∶0)。此外,对菌丝中杀虫相关的环肽beauverolides,beauvericins和bassianolide进行相对含量比较分析,发现单独一种肽的含量高低与菌株对茶毛虫的毒力没有直接关系,但高毒力的Bb1898菌株中的9种肽同时具有较高的含量暗示它们可能发挥协同作用。【结论】毒力和萌发的共同标记物是脂酰肉碱和磷脂,它们可能具有维持附着胞膨压和为穿透寄主提供能量的功能。其它标记物,如在高毒力菌丝中发现的hercynine和α,α-trehalose;高萌发率孢子中的2,3-dimethylmaleate,高毒力孢子中的histamine和2,5-pyrrolidinedicarboxylic acid Diamide,它们的作用原理有待进一步研究。     

Effect of nutrition on growth and virulence of the entomopathogenic fungus Beauveria bassiana

Three isolates of the entomopathogen Beauveria bassiana along with one strain of Metarhizium anisopliae were cultured on seven media with different carbon/nitrogen (C/N) ratios. The effect of nutrition on virulence of the isolates was evaluated via measurement of colony growth, spore yield, germination speed, conidial C/N ratio and Pr1 (a serine protease) activity. 'Osmotic stress' medium produced the lowest colony growth with low numbers of conidia in all isolates. However, these conidia showed a high germination rate and virulence. However, conidial Pr1 activity was low in some isolates. In most but not in all cases conidia from 1% yeast extract, 2% peptone and low (10 : 1) C/N medium had higher Pr1 activity compared with conidia from other media. However, in some instances we could not conclude that there was a relationship among germination rate, conidial Pr1 activity and virulence. C/N ratio of conidia was statistically different among various media and fungal isolates. Conidia with lower C/N ratio generally produced lower LT(50) (lowest median lethal time) values (more virulent). Insect-passaged conidia from different media had lower C/N ratio compared with similar conidia from artificial cultures. Therefore, they should be more virulent than in vitro produced conidia. As germination rate, conidial Pr1 activity and C/N ratio are independent of host, it seems that host-related determinants such as insect cuticle and physiology and environmental conditions may influence host susceptibility and therefore fungal isolate virulence towards host insects.     

A cuticle-degrading protease (CDEP-1) of Beauveria bassiana enhances virulence

In order to investigate virulence enhancement of entomopathogenic fungi, a Beauveria bassiana-sourced Pr1 protease (CDEP-1) was expressed by a methylotrophic yeast Pichia pastoris and then used as an additive to three gradient sprays of B. bassiana strain (Bb0062) onto apterous green peach aphid Myzus persicae adults in six bioassays. The resultant data fit well to a time–concentration–mortality model. Generally, the LC₅₀ estimates of the fungal pathogen against the aphid species decreased with increasing CDEP-1 concentrations from 0 to 100 µg mL^?1. The LC₅₀s on days 5–7 after spray were reduced by 1.5–2.5-fold at the concentrations of 20–100 µg mL^?1. However, sprays of 20–100 µg CDEP-1 mL^?1 aqueous solution alone had no significant effect on aphid mortality compared to water spray only. Neither did inclusion of inactivated CDEP-1 at a concentration of 50 µg mL^?1 affect significantly the fungal virulence to aphids. Our results confirm for the first time that the cuticle-degrading protease CDEP-1 enhanced fungal virulence due to acceleration of conidial germination and cuticle penetration. This suggests a new approach to utilising the protease in microbial control.