红火蚁Serpin家族基因鉴定及其在绿僵菌侵染下的表达模式分析

丝氨酸蛋白酶抑制剂（Serine protease inhibitor, Serpin）在昆虫Toll通路和PPO级联反应的防御机制中起着至关重要的作用。本研究利用现代生物信息学工具预测和分析了红火蚁Solenopsis invicta中Serpin家族的结构和功能多样性。染色体定位表明,红火蚁基因组中7个Serpin基因家族成员不均匀的分布在4条染色体上。结构域分析表明,SiSerpin蛋白具有Serpin基因的保守结构,即典型的反应中心环（Reactive central loop, RCL）结构与Serpin结构域,不同成员蛋白拥有多样化的基序。系统进化分析表明,红火蚁Serpin蛋白与其他的昆虫Serpin蛋白有较高亲缘关系,SiSerpin7与烟草天蛾Manduca sexta的Serpin3a亲缘关系最近,SiSerpin12与黄粉虫Tenebrio molitor Serpin55亲缘关系最近,SiSerpin13与烟草天蛾Serpin6亲缘关系最近,SiSerpin4与家蚕Serpin27亲缘关系最近。为了进一步明确红火蚁Serpin家族在绿僵菌Metarhizium侵染后的表达模式,用绿僵菌侵染不同体型的工蚁,荧光定量PCR（RT-qPCR）分析表明,红火蚁Serpin基因家族成员能在早期响应绿僵菌的侵染;除SiSerpin7基因外,大型和小型两种工蚁的表达模式存在较大的差异。结果表明红火蚁Serpin家族作为免疫调控蛋白,能够响应绿僵菌的侵染,且在红火蚁不同体型工蚁之间存在着不同的免疫调控模式。这些发现不仅为Serpins的功能分析提供了理论基础,而且有可能有助于绿僵菌作为一种有效的生防剂的开发。     

实时荧光定量PCR检测感病蝗虫体内绿僵菌rDNA基因

根据绿僵菌23SrDNA的转录间隔区(ITS)和5.8S的基因序列,设计检测感病蝗虫体内绿僵菌菌体DNA分子的特异引物,通过优化感病蝗虫菌体DNA快速制备方法,建立了基于荧光定量PCR的定量检测体系。该检测方法专一、灵敏,检测下限为10pg/μl绿僵菌,并且能从刚侵染48h的东亚飞蝗血淋巴中,早期定量检测到在虫体血淋巴中增殖的绿僵菌的rDNA。     

飞蝗解毒酶系活力测定方法

飞蝗Locusta migratoria是重要的农业害虫,代谢抗性是飞蝗主要的农药抗性机制之一。与代谢抗性相关的解毒酶系主要有:非专一性酯酶系(Non-specficesterases,ESTs)、谷胱甘肽S-转移酶系(Glutathione S-transferases,GSTs)和细胞色素P450单加氧酶系(Cytochrome P450 monooxygenases,P450s),解毒酶系活力的测定是研究飞蝗农药代谢机制的重要途径。本文详细介绍了飞蝗解毒酶系的测定方法,为蝗虫及其他昆虫解毒酶系的测定提供参考。     

曲酸对飞蝗酚氧化酶以及其他生化酶活性的影响

酚氧化酶(Phenoloxidase)可以催化黑色素中间体多巴胺的形成,是昆虫黑化作用中的关键酶,在昆虫细胞免疫和体液免疫过程中发挥着重要的作用。"近年来,酚氧化酶成为新型杀虫剂靶标的研究对象之一,而酚氧化酶的抑制剂曲酸(Kojic acid)对昆虫解毒酶和保护酶的影响的研究却很少。为了探索酚氧化酶与昆虫其他解毒酶或保护酶类的关系,本文利用含不同剂量曲酸的麦麸饲喂飞蝗,检测飞蝗存活率以及虫体酚氧化酶(PO)、乙酰胆碱酯酶(AChE)、解毒酶(ESTs和GSTs)和保护酶(POD、SOD和CAT)的活性。结果发现,曲酸对飞蝗无明显毒力;在曲酸处理1 d和3 d后,曲酸处理组飞蝗PO二酚酶活性显著低于对照,5 d和7 d后飞蝗PO活性与对照相比差异不大;处理3 d后,飞蝗AChE活性显著低于对照,而5 d后AChE相比于对照显著升高;偏高浓度的曲酸在1 d和3 d后对飞蝗ESTS和GSTs有显著的抑制,5 d和7 d后飞蝗ESTs和GSTs活性上升;曲酸在1 d和3 d后引起了飞蝗保护酶的升高,5 d后飞蝗保护酶有一定的下降。以上结果表明,曲酸对飞蝗PO有明显抑制作用,同时也干扰了昆虫其它生化酶的活性。由此推测,曲酸可以作为杀虫剂或者杀虫真菌的增效剂。     

玫烟色棒束孢侵染对小菜蛾幼虫体内不同酶活的影响

在实验室条件下,测定了昆虫病原真菌玫烟色棒束孢Isaria fumosorosea的侵染对小菜蛾Plutella xylostella3龄幼虫体内酚氧化酶(PO)和不同抗氧化酶类,包括超氧化物歧化酶(SOD)、过氧化物酶(POD)、过氧化氢(CAT)及谷胱甘肽S-转移酶(GSTs)活力的变化。结果表明,玫烟色棒束孢侵染小菜蛾3龄幼虫后,体内PO、SOD、POD、CAT和GSTs活力均受到明显的影响。其中感病小菜蛾幼虫体内的PO活力始终高于同期未感染的小菜蛾幼虫,当接菌40h时,酚氧化酶活力达到最大37.4U/g,为对照的2.6倍,而SOD、POD、CAT和GSTs活力在感病前期明显高于对照,接菌40–48h各种酶活力均达到最大,当接菌56h酶活力开始下降,64h时酶活力均明显低于对照。可见玫烟色棒束孢的侵染严重干扰了小菜蛾幼虫体内正常的生理活动。     

亚致死质量浓度联苯肼酯对截形叶螨解毒酶系的影响

【目的】明确联苯肼酯的亚致死质量浓度对截形叶螨Tetranychus truncatus解毒酶系的影响,从而为进一步揭示截形叶螨的代谢抗性及联苯肼酯的科学使用提供理论依据和指导。【方法】采用叶片浸渍法测定了联苯肼酯对截形叶螨雌成螨的LC50,应用毒力回归方程计算截形叶螨雌成螨死亡率为10%和30%时联苯肼酯的亚致死质量浓度LC10和LC30,并处理截形叶螨雌成螨,以甲醇水溶液作为对照,测定并分析其体内羧酸酯酶(CarE)、谷胱甘肽S-转移酶(GSTs)、多功能氧化酶(MFO)的比活力及酶动力学常数(米氏常数Km和最大反应速率Vmax)的变化。【结果】比活力与对照相比,LC10、LC30处理后,截形叶螨雌成螨体内CarE、GSTs和MFO的比活力均显著升高(P0.05),且LC30处理组CarE、GSTs(除24 h外)比活力显著高于LC10(P0.05)。酶动力学常数:体内CarE的Km显著减小或无明显变化(P0.05),Vmax显著增大(P0.05),即CarE与底物亲和力增大,反应速率加快;而GSTs和MFO的Km显著增大或无明显变化(P0.05),Vmax均显著减小(P0.05),说明GSTs和MFO与底物亲和力低,反应速率降低。【结论】联苯肼酯对截形叶螨雌成螨体内的解毒酶系有诱导作用,CarE在截形叶螨对该杀螨剂的代谢中起主导作用。     

蝗绿僵菌氰化物水合酶基因MaChy的克隆及表达分析

采用筛选cDNA文库的方法,首次克隆了蝗绿僵菌氰化物水合酶基因MaChy的全长cDNA序列和DNA序列,序列分析表明,蝗绿僵菌氰化物水合酶基因MaChy不含内含子,开放阅读框（ORF）为1,074bp,编码357个氨基酸,推测蛋白的分子量为39.78kDa,等电点（pI）为5.53;利用在线软件分析表明,该蛋白既不是分泌型蛋白也不是膜蛋白;同源性比对结果表明,该蛋白与其他真菌中的氰化物水合酶蛋白的同源性较高,具有高度保守的催化三联体特征。用qRT-PCR方法分析了该基因在蝗绿僵菌侵染昆虫过程中的表达情况,结果表明,MaChy在蝗绿僵菌侵染穿透昆虫体壁阶段的表达量最高,约为体内阶段表达量的2.5倍,推测该基因可能在蝗绿僵菌穿透昆虫体壁的过程中起重要作用。     

辣椒素对西花蓟马雌成虫体内解毒酶活性的影响

【目的】为明确非挥发性次生物质辣椒素对西花蓟马Frankliniella occidentalis雌成虫体内解毒酶活性的影响。【方法】将菜豆叶片研碎离心后取上清液作为西花蓟马的液态饲料,将不同浓度的辣椒素添加到饲料中,饲养西花蓟马雌成虫48、72、96 h后,分别测定西花蓟马体内羧酸酯酶(Car E)、谷胱甘肽-S-转移酶(GSTs)、乙酰胆碱酯酶(AChE)和多功能氧化酶(MFO)的活性。【结果】低剂量辣椒素能诱导西花蓟马雌成虫体内GSTs、AChE和MFO活性明显升高,高剂量辣椒素抑制GSTs、AChE及MFO活性。不论在哪一种辣椒素浓度下,Car E活性一直受到抑制。辣椒素浓度和处理时间对GSTs及AChE活性无明显交互作用,但对Car E及MFO有明显的交互作用,处理时间越长,作用越显著。【结论】西花蓟马雌成虫体内解毒酶活性在次生物质辣椒素处理下发生了明显的变化,说明西花蓟马通过解毒酶适应次生物质对其的影响,但解毒酶的变化有剂量和时间效应。     

Cry1Ac杀虫蛋白对粘虫中肠几种酶活性的影响

为阐明Bt杀虫蛋白对次要靶标害虫粘虫Mythimna separata (Walker) （鳞翅目： 夜蛾科）的生理学影响, 本研究分析比较了粘虫高龄幼虫在室内取食低剂量Cry1Ac杀虫蛋白6, 12, 24和36 h后, 其体内主要的解毒酶（酯酶和谷胱甘肽-S-转移酶）、 保护酶（超氧化物歧化酶、 过氧化氢酶和过氧化物酶）和中肠蛋白酶（总蛋白酶、 强碱性类胰蛋白酶、 弱碱性类胰蛋白酶和类胰凝乳蛋白酶）等活性的变化。结果表明, 取食Cry1Ac杀虫蛋白后, 粘虫幼虫体内相关酶活力呈现不同的变化趋势： （1）酯酶、 谷胱甘肽-S-转移酶、 过氧化物酶(POD)、 类胰蛋白酶和类胰凝乳蛋白酶活力较对照显著降低（P<0.05）; （2）超氧化物歧化酶(SOD) 活力较对照显著升高（P<0.05）; （3）过氧化氢酶(CAT) 活力于6, 12和24 h显著低于对照（P<0.05）, 36 h时显著高于对照（P<0.05）。结果提示Cry1Ac杀虫蛋白主要通过抑制粘虫幼虫中肠解毒酶和蛋白酶的活性, 扰乱SOD, CAT 和POD 3种保护酶的动态平衡而干扰幼虫的正常生理代谢, 从而起到毒杀粘虫的作用。     

昆虫病原微生物及其在蝗灾治理中的应用

本文重点介绍了蝗虫痘病毒、蝗虫微孢子虫、蝗噬虫霉、绿僵菌、白僵菌、米曲霉等的生物学特性、侵染机制、流行病学和应用潜力。蝗虫痘病毒和蝗噬虫霉分别由于生产成本过高和分生孢子在离体条件下存活时间过短而难以开发应用;蝗虫微孢子虫和绿僵菌已广泛应用到蝗灾治理中,有关绿僵菌致病机理的研究最深入全面;白僵菌也已成功应用到蝗灾治理中;介绍了一种新病原——米曲霉对飞蝗的毒力以及产孢量和耐热性等生物学特性。最后,提出蝗虫新病原微生物资源的开发、提高生产工艺水平、研发新的制剂和延长储存时间等是今后蝗虫微生物农药的发展方向。     

金龟子绿僵菌IMI330189液体发酵动力学研究

研究了金龟子绿僵菌IMI330189的液体发酵动力学。利用Sigmoid函数构建了该菌株液体发酵过程中的菌体生长和底物消耗的动力学模型,并运用Origin7.5软件拟合求解出各模型参数。结果表明,模型能够较好地拟合绿僵菌IMI330189液体发酵过程,其比生长速率在发酵第22.8h达到最大值,为0.084h-1;总糖比消耗速率在第9.6h达到最大值,为0.246h-1;总氮比消耗速率在第10.3h达到最大值,为0.007h-1;菌体对总糖的得率系数在39.8h达到最高,为0.861g/g。模型拟合和实验数据具有良好的适应性,基本反映了绿僵菌IMI330189液体发酵过程的动力学特征,为其液体发酵工艺的优化和发展奠定了基础。     

Trehalose and trehalose-hydrolyzing enzyme in the haemolymph of Locusta migratoria infected with Metarhizium anisopliae strain CQMa102

Topical application of the Metarhizium anisopliae var. acridum specialist strain CQMa 102 to the locust Locusta migratoria manilensis results in changes of the concentrations of trehalose and glucose in the haemolymph. Micrographs of the locust haemolymph shows Metarhizium anisopliae can effectivly penetrate the external skeleton of locust and after 2 days infection, the hyphae body will appear in the haemolymph of infected insects. The time in decrease of trehalose concentration coincided with that in increase of trehalose-hydrolysing enzyme activity in the haemolymph of the fungus-infected insects. Overlay gel analysis indicated there was considerably more trehalose-hydrolysing activity in the haemolymph of locusts infected by fungus than in controls. A comparable isoform was identified in in vitro culture of the fungus, suggesting a fungal origin for the in vivo enzyme. Haemolymph trehalose decreased significantly during mycosis of locusts by M. anisopliae. All these results suggested that this fungus may take advantage of competing nutrient utilization against the insect by its trehalose-hydrolyzing enzyme secretion. It may provide fundamental knowledge for fungal pathogenesis.     

东亚飞蝗UBX结构域包含蛋白基因 LmUBX2 的克隆和表达分析

【目的】UBX结构域包含蛋白是p97/CDC48的辅助因子。p97在泛素化相关的多种细胞过程中起着重要的作用,如依赖泛素 蛋白酶体系统的蛋白质降解和同型膜融合等。本研究旨在克隆东亚飞蝗 Locusta migratoria manilensis (Meyen)的UBX结构域包含蛋白基因,分析其组织和发育表达格局,为进一步研究UBX结构域包含蛋白基因的功能奠定基础。【方法】通过分析东亚飞蝗的转录组数据克隆UBX结构域包含蛋白基因,采用实时定量PCR技术分析该基因在不同发育时期和成虫不同组织中的表达水平。【结果】克隆到东亚飞蝗的一个UBX结构域包含蛋白基因,命名为 LmUBX2。 LmUBX2 开放阅读框长1 020 bp,编码399个氨基酸,预测分子量和等电点分别为37.8 kDa和6.03,与其他UBX结构域包含蛋白的氨基酸一致性为37%~64%,N端和C端分别有一个保守的UBA结构域和UBX结构域。序列比较和系统发育分析发现 LmUBX2 属于SAKS1亚家族。定量分析发现,LmUBX2 在整个生命周期中都有表达,但成虫期的表达水平最高;在检测的所有组织中都有表达,但在精巢和卵巢中表达水平最高。【结论】研究结果说明 LmUBX2 可能参与东亚飞蝗多种生理过程,尤其可能与东亚飞蝗的生殖有关,但还需深入研究。     

Cloning of a novel protease required for the molting of Locusta migratoria manilensis

Molting is required for progression between larval stages in the life cycle of an insect. The essence of insect molting is the laying down of new cuticle followed by shedding of the old cuticle. Degradation and recycling of old cuticle are brought about by enzymes present in the molting fluid, which fills the space between the old and new cuticle. Here, we describe the cloning of a novel protease gene from Locusta migratoria manilensis, designated as Lm-TSP. The cDNA and its deduced protein sequences were deposited in GenBank (accession numbers EF081255 and ABN13876, respectively). Sequence analysis indicated that Lm-TSP belongs to the trypsin-like serine protease family. We show, by RNA interference (RNAi), that silencing of Lm-TSP leads to dramatic reductions in protease and cuticle-degrading activity of a molting fluid, which leads to molting defects from fourth-instar larvae (L4) to fifth-instar larvae (L5), and between L5 and adult stages. These observations suggest that Lm-TSP plays a critical role in L. migratoria manilensis ecdysis.     

Heterologous expression and characterization of a sigma glutathione S-transferase involved in carbaryl detoxification from oriental migratory locust, Locusta migratoria manilensis (Meyen)

Glutathione S-transferases (GSTs) play a major role in detoxification of xenobiotics and resistance to insecticides in insects. In the present study, a sigma-class GST gene (LmGSTs3) was identified from the locust, Locusta migratoria manilensis. Its full-length cDNA sequence is 828 bp containing an open reading frame (ORF) of 612 bp that encodes 204 amino acid residues. The predicted protein molecular mass and pI are 23.4 kDa and 7.62, respectively. Recombinant LmGSTs3 was heterologously expressed in Escherichia coli as a soluble fusion protein. Its optimal activity was observed at pH 8.0. Incubation for 30 min at temperatures below 40 °C scarcely affected activity. The LmGSTs3 at pH values between 4.0 and 11.0 retained more than 80% of its original activity. Ethacrynic acid and cibacron blue were very effective inhibitors of LmGSTs3 with I50-values 1.7 and 3.7 μM, respectively. In response to heavy metal (CuSO4, CdCl2) exposure there was a concentration-dependent and time-dependent decrease in activity. The nymph mortalities after carbaryl treatment increased 38.7% after LmGSTs3 were silenced. These results suggest that LmGSTs3 may be involved in carbaryl detoxification in L. migratoria manilensis.     

东亚飞蝗感染绿僵菌后的组织病理变化

将人工培养的绿僵菌分生孢子配制成浓度为 1× 1 0 8分生孢子 mL的孢子油剂 ,涂抹在供试东亚飞蝗Locustamigratoriamanilensis(Meyen)幼虫的腹侧部。通过组织切片研究表明 ,绿僵菌主要是从东亚飞蝗体表侵入的 ,72h后可见体腔内有菌丝和组织病变 ,侵入体内的菌丝在血腔中不断增殖 ,使得脂肪体、肌肉组织、马氏管和消化道发生病变解体。 96h后 ,肠壁细胞结构开始疏松、内膜解体。 1 2 0h后 ,多数幼虫死亡 ,消化道内外布满菌丝。     

Optimization of parental RNAi conditions for hunchback gene in Locusta migratoria manilensis (Meyen)

An investigation on the optimization of parental RNA interference (RNAi) conditionsfor hunchback {hb) gene in Locusta migratoria manilensis (Meyen) was conducted.Double stranded RNA (dsRNA) corresponding to hb gene was injected into haemocoel offemale adults of L.migratoria manilensis.Embryos developed from the eggs laid by theinjected adults on the 7th day after eclosion showed observable effects of RNAi for hb.Thesilencing effect after delivery treatment of dsRNA for hb gene was maintained for moretha...     

In vivo study on combined toxicity of Metarhizium anisopliae (Deuteromycotina: Hyphomycetes) strain ESC-1 with sublethal doses of chlorpyrifos, propetamphos, and cyfluthrin against German cockroach (Dictyoptera: Blattellidae)

The effect of Metarhizium anisopliae (Metschnikoff) Sorokin strain ESC-1 alone and in combination with sublethal doses of commercial formulations of chlorpyrifos, propetamphos and cyfluthrin on mortality of CSMA strain of German cockroach, Blattella germanica (L.), was determined by conducting in vivo studies that included 3 bioassays. Spores of M. anisopliae cultured on SDAY media had germination of >90%. Based on bioassay 1, doses ranging from 0.5 to 300 ppm of chlorpyrifos and propetamphos, and 0.05 to 40 ppm of cyfluthrin were selected for bioassays 2 and 3. Cockroach mortality ranged from 5 to 20% for insecticides alone and 48 to 70% for insecticides + M. anisopliae in bioassay 2. In bioassay 3, mortality ranged from 15 to 60% for insecticides and 57.5 to 92.5% for insecticides + M. anisopliae. Percentage of cockroach mortality resulting from insecticide + M. anisopliae combinations was significantly higher than insecticide alone. Mortality was also significantly higher in certain insecticide + M. anisopliae combinations than M. anisopliae alone. There was no significant interaction between M. anisopliae and insecticides with their concentrations in bioassay 2, indicating an additive effect. But in bioassay 3, a significant interaction was observed when M. anisopliae was combined with multiple insecticide concentrations. The interaction indicated an additive effect for chlorpyrifos and cyfluthrin, and a synergistic effect for propetamphos. There were significant differences in LT50 among various treatment combinations. M. anisopliae alone or insecticide + M. anisopliae combinations did not affect body weight in treated German cockroaches.