斯氏按蚊中Toll受体参与抵抗微生物感染和调控肠道菌群稳态

【目的】Toll信号通路是昆虫天然免疫系统的重要组分,其中Toll受体在激活昆虫病原菌侵染免疫应答方面发挥了关键作用。本研究旨在探究斯氏按蚊Anopheles stephensi Toll受体基因在抵抗微生物侵染和维持肠道菌群稳态过程中的功能。【方法】根据冈比亚按蚊Anopheles gambiae Toll受体家族的蛋白氨基酸序列,通过序列同源比对鉴定斯氏按蚊中相应的Toll受体基因;运用荧光定量PCR检测Toll受体基因在未感染病原菌的斯氏按蚊脂肪体中的相对表达量,以及在真菌球孢白僵菌Beauveria bassiana和革兰氏阴性细菌胡萝卜软腐欧文氏菌Erwinia carotovora subsp. carotovora侵染斯氏按蚊过程中的表达变化;最后,在斯氏按蚊雌成蚊胸部显微注射AsToll1A和AsToll5A的双链RNA进行RNA干扰后,检测RNAi处理的斯氏按蚊受真菌侵染后的存活率、肠道细菌含量变化以及抗菌肽基因表达变化。【结果】在斯氏按蚊中共鉴定到8个Toll受体基因,即AsToll1A, AsToll5A, AsToll6, AsToll7, AsToll8, AsToll9, AsToll10和AsToll11。通过荧光定量PCR检测发现,未感染病原菌的斯氏按蚊雌成蚊脂肪体中AsToll5A表达量最高,AsToll1A表达量次之,其余Toll受体基因表达量极低。在球孢白僵菌和胡萝卜软腐欧文氏菌侵染过程中,与对照(注射PBS)比较,AsToll1A和AsToll5A在斯氏按蚊中的表达量显著升高,其余Toll受体基因表达变化不显著或降低。RNA干扰结果表明,AsToll1A或AsToll5A的表达受到抑制后,斯氏按蚊对球孢白僵菌的抵抗能力显著降低,肠道细菌总量与对照(dsGFP)比较显著增多。而且,抑制AsToll1A后抗菌肽基因DEF1和GAM1的表达受到显著抑制;抑制AsToll5A后仅有GAM1表达量下调。【结论】斯氏按蚊Toll受体在结构和功能上具有高度的保守性,其中AsToll1A和AsToll5A能响应病原真菌和革兰氏阴性细菌侵染并且影响肠道菌稳态。     

孝顺竹肉桂醇脱氢酶基因的克隆及表达研究

肉桂醇脱氢酶(cinnamoyl alcohol dehydrogenas,CAD)是木质素生物合成过程中的一类关键酶.采用RT-PCR及RACE方法从孝顺竹笋中分离出CAD基因家族的一个基因,cDNA全长是1131 bp(GenBank注册号为GU985522),含有一个1 107 bp的读码框,编码一个368 aa...     

冈比亚按蚊性别决定基因 doublesex 的克隆、序列分析及表达谱

【目的】doublesex 是控制昆虫性别分化的关键基因,决定了昆虫体细胞与生殖细胞的性别。本研究旨在克隆、鉴定重要疟疾媒介冈比亚按蚊 Anopheles gambiae 性别决定基因 doublesex（Angdsx）,分析其在雌雄个体内的剪切体及在不同发育时期的表达模式。【方法】基于冈比亚按蚊转录组数据库,比对到 Angdsx 相关片段,分别以雌雄成蚊cDNA为模板,采用RT-PCR与RACE方法克隆分别获得雌雄个体内 Angdsx 全长基因,利用生物信息软件对所得序列进行结构域预测、氨基酸序列比对和进化树分析。根据 Angdsx 特异性表达引物,利用RT-PCR方法研究其在冈比亚按蚊雌雄个体及不同发育时期的表达谱。【结果】分别从冈比亚按蚊雌雄成虫中克隆获得 Angdsx cDNA全长序列,分别命名为Angdsx^F（GenBank登录号：KM978937）和 Angdsx ^M（GenBank登录号：KM978938）。Angdsx 位于2号常染色体右臂,基因横跨接近80 kb基因组长度。Angdsx^F 长度为4 874 nt,编码长度为265 个氨基酸的雌性特异性蛋白DSX^F;Angdsx ^M 长度为3 183 nt,编码长度为633个氨基酸的雄性特异性蛋白DSX^M。结构域分析发现 Angdsx 包括 doublesex 保守的TRA/TRA-2结合位点、dsx 重复序列、富含精氨酸/丝氨酸双肽区、多聚嘌呤增强子序列和RNA结合蛋白结合序列,以及连续的双核苷酸GT为主的重复序列。与Angdsx^F 相比, Angdsx ^M具有一个雌性特异性的外显子。Angdsx ^M 在0-2 h卵中高表达,随后逐渐减少,在12-24 h卵中降至最低,之后再次升高;Angdsx^F 则在6-8 h卵中开始表达。【结论】本研究获得了冈比亚按蚊性别决定基因 Angdsx 在雌雄个体内的全长序列,Angdsx 具有保守的结构域与表达特征。本研究结果为蚊虫性别分化的分子机制及将其最终应用于显性致死昆虫施放技术进行蚊媒的防制提供了理论基础。     

按蚊防御素基因家族转录模式和启动子的生物信息学分析

运用生物信息学方法分析冈比亚按（Anopheles gambiae）中防御素基因家族的表达模式和启动子保守区。在ENSEMBL数据库中搜索防御素基因家族序列,然后将基因编号输入angaGEDUCI数据库中进行分析。结果在冈比亚按蚊中存在有4个防御素基因的异构体分子,其中DEF1基因表达模式与其它3个基因存在明显差异。4个防御素基因启动子序列都存在AP-1、GATA-1、GCN4、GR、HNF-3B、TFIID6个转录因子的识别结合位点,表明这6个转录因子是冈比亚按蚊防御素基因家族表达调控所必需的。值得注意的是,DEF1基因启动子序列中存在与性别决定翦关的SOXproteins转录因子的结合位点,暗示DEF1基因的转录调控可能与性别分化有关;而在DEF2基因启动子序列中存在有热激因子（HSTF）的识别结合位点,表明热刺激会调控DEF2基因的表达。     

斯氏按蚊泛素羧端水解酶对约氏疟原虫感染应答性表达增高(英文)

分离和研究疟疾感染蚊的差异表达基因 ,对阐明媒介与疟原虫之间相互作用及其分子机制尤为重要。利用已建立的斯氏按蚊感染约氏疟原虫的差减cDNA库的进行表达筛选 ,发现表达增高基因中有一个编码与黑腹果蝇泛素羧端水解酶高度同源蛋白的序列。相似性比较显示该编码序列在氨基酸水平与已知的冈比亚按蚊EST序列对应部位的同源性为 89% ,与果蝇和人类的同源性均为 63%。模拟Northern印迹的表达动态分析提示 ,感染后至少 1～ 7天内该基因在蚊体内的表达显著增高 ,与疟原虫发育动合子穿越蚊中肠壁和子孢子从卵囊向蚊眼涎腺移行等关键阶段相一致。目前对有关蚊天然免疫系统激活的泛素途径所知甚少 ,现有结果提示该基因与疟原虫感染相关 ,它的克隆和表达分析有可能推测其在疟原虫感染中所起的作用     

斯氏按蚊泛素羧端水解酶对药氏疟原虫感染应答性表达增高

分离和研究疟疾感染蚊的差异表达基因,对阐明媒介与疟原虫之间相互作用及其分子机制尤为重要,利用已建立的斯氏按蚊感染约氏疟原虫的差减cDNA库的进行表达筛选,发现表达增高基因中有一个编码与黑腹果蝇泛素羧端水解酶高度同源蛋白的序列,相似性比较显示该编码序列在氨基酸水平与已知的冈比亚按蚊EST序列对应部位的同源性为89％,与果蝇和人类的同源性均为63％,模拟Northern印迹的表达动态分析提示,感染后至少1－7天内该基因在蚊体内的表达显增高,与疟原虫发育动合子穿越蚊中肠壁和子孢子从卵囊蚊眼涎腺移行等关键阶段相一致。目前对有关蚊天然免疫系统激活的泛素途径所知甚少,现有结果提示该基因与疟原虫感染相关,它的克隆和表达分析有可能推测其在疟原虫感染中所起的作用。     

林氏按蚊线粒体全基因组序列的测定及基于线粒体基因组的按蚊属系统发育分析(英文)

【目的】对林氏按蚊Anopheles lindesayi完整的线粒体基因组进行测序及分析,依据已知的线粒体基因组构建并讨论按蚊属蚊虫的分子系统发育关系。【方法】对林氏按蚊线粒体基因组进行测序、注释,并对其基本特征和基本组成进行分析。基于串联的13个蛋白质编码基因的核苷酸序列和氨基酸序列,用ML法和贝叶斯法构建林氏按蚊和按蚊属其他32种蚊虫的系统发育树,据此探讨按蚊属蚊虫的系统发育关系和系统分类。【结果】林氏按蚊线粒体基因组全长为15 366 bp,包含13个蛋白质编码基因,22个tRNA基因,2个rRNA基因和一段控制区。林氏按蚊线粒体基因组呈现明显的AT偏斜和GC偏斜,AT偏斜为正,GC偏斜为负。除了COX1使用TCG和ND5使用GTG作为起始密码子以外,其他蛋白质编码基因的起始密码子均遵循ATN原则;终止密码子为TAA或者T。除了tRNA^Ser(AGN)以外,其他的tRNA基因均呈现典型的三叶草二级结构。控制区AT含量最高,为94.54%。滑窗分析显示蛋白质编码基因是用于构建亚属或属水平系统发育关系的最佳分子标记。系统发育树强烈支持塞蚊亚属Cellia、按蚊亚属Anopheles、徕蚊亚属Nyssorhynchus和柯特蚊亚属Kerteszia均为单系群。小五斑按蚊An. atroparvus和四斑按蚊An. quadrimaculatus A这两个种聚到一起,从传统的形态分类上讲,它们和林氏按蚊均属于按蚊亚属按蚊系蚊虫。但本研究构建的4个系统发育树均显示,(小五斑按蚊An. atroparvus+四斑按蚊An. quadrimaculatus A)和林氏按蚊被属于迈蚊系的中华按蚊分开,这为两个系的分类提供了新的论点。【结论】本研究获得了林氏按蚊的完整的线粒体基因组,探析了按蚊属的线粒体基因组特征和系统发育关系,为进一步研究蚊科线粒体基因组和系统发育关系提供了依据。     

傅氏按蚊与朝鲜按蚊的形态对比观察

傅氏按蚊(Anopheles freyi Meng,1957)和朝鲜按蚊(An.koreicus Yamada and Watanabe,1918)形态十分相似,二者是独立的种还是同种异名,迄今仍有争议〔1〕,我们用形态特征结合雄蚊抱握器运动频率进行了比较观察,报告如下。1材料与方法傅氏按蚊捕自雅安地区名山县车岭乡牛舍,雌蚊单个鉴定,产卵,培育后代。对成虫观察形态,并按康万民等〔2〕和潘家复等〔3〕方法观察抱握器运动频率。共观察32只,平均69.44±1.48次。朝鲜按蚊形态以冯兰洲〔4〕、陆宝麟〔5〕和雷心田〔1〕描述的形态特征为依据。2结果傅氏按蚊和朝鲜按蚊的翅上白斑、黑斑、抱握…     

印度疟疾媒介库态按蚊卵黄蛋白原基因的克隆与生物信息学分析（英文）

卵黄蛋白原（vitellogenin, Vg)是主要的卵黄蛋白前体, 在雌虫血餐之后在脂肪体内大量合成。卵黄蛋白原的调节元件已经被用于驱动蚊子（与寄生虫发生最大相互作用的场所）中抗寄生基因的组织特异性表达。不过, 迄今为止, 对在印度引起60%~70%疟疾发生的库态按蚊Anopheles culicifacies中的内源启动子尚未进行过分析。本研究通过PCR扩增了包括5′端上游调节区在内的库态按蚊A. culicifacies卵黄蛋白原基因, 并命名为AncuVg (GenBank登录号为JN113091)。它含有一个大约6.2 kb的开放阅读框, 编码2 052个氨基酸, 具有一个16个氨基酸残基的推断的信号肽。也含有一个N_Vitellogenin区和一个VWF型D区, 这两个区在其他昆虫卵黄蛋白原中也保守。估计多肽分子量为238.0 kDa, 含有4个共有的(RXXR/S)切割位点, C端附近有一个GL/ICG基序, 其后是9个半胱氨酸残基和1个位于GL/ICCG基序上游第18个氨基酸残基处的DGXR 基序。在推断的氨基酸序列上发现3个聚丝氨酸区, 其中2个位于氨基端, 1个位于羧基端。根据同义密码子相对使用概率值, 通过有效密码子数, 测定了蚊子卵黄蛋白原基因密码子的偏倚性程度。也预测了库态按蚊A. culicifacies Vg的三维结构。分析了AncuVg基因, 以理解Vg基因的转录调节。对Vg基因5′端上游区进行的系统发育分析表明, 它们聚类于蚊子的3大分枝。也用各种生物信息学工具分析分析了Vg的同源性和特征。     

淡色库蚊抗药性相关胰蛋白酶基因cDNA全长克隆与序列分析

用逆Northern印迹和Northern印迹法进一步鉴定淡色库蚊对溴氰菊酯抗药性和敏感性品系胰蛋白酶的表达差异 ,结果显示 ,胰蛋白酶基因在抗药性品系中的表达量分别是敏感性品系的 4.3和 3.9倍。采用RACE法筛选cDNA文库 ,获得总长度为 90 9bp的淡色库蚊胰蛋白酶基因的全长序列 ,其中开放阅读框为 786bp ,推导出编码 2 6 1个氨基酸的蛋白质 (GenBank/NCBIAY0 34 0 6 0 ) ,其与冈比亚按蚊胰蛋白酶同源性最高 ,为 5 5 %     

Inducible expression of double-stranded RNA reveals a role for dFADD in the regulation of the antibacterial response in Drosophila adults

In Drosophila, the immune deficiency (Imd) pathway controls antibacterial peptide gene expression in the fat body in response to Gram-negative bacterial infection. The ultimate target of the Imd pathway is Relish, a transactivator related to mammalian P105 and P100 NF-kappaB precursors. Relish is processed in order to translocate to the nucleus, and this cleavage is dependent on both Dredd, an apical caspase related to caspase-8 of mammals, and the fly Ikappa-B kinase complex (dmIKK). dTAK1, a MAPKKK, functions upstream of the dmIKK complex and downstream of Imd, a protein with a death domain similar to that of mammalian receptor interacting protein (RIP). Finally, the peptidoglycan recognition protein-LC (PGRP-LC) acts upstream of Imd and probably functions as a receptor for the Imd pathway. Using inducible expression of dFADD double-stranded RNA, we demonstrate that dFADD is a novel component of the Imd pathway: dFADD double-stranded RNA expression reduces the induction of antibacterial peptide-encoding genes after infection and renders the fly susceptible to Gram-negative bacterial infection. Epistatic studies indicate that dFADD acts between Imd and Dredd. Our results reinforce the parallels between the Imd and the TNF-R1 pathways.     

Peptidoglycan molecular requirements allowing detection by the Drosophila immune deficiency pathway

Innate immune recognition of microbes is a complex process that can be influenced by both the host and the microbe. Drosophila uses two distinct immune signaling pathways, the Toll and immune deficiency (Imd) pathways, to respond to different classes of microbes. The Toll pathway is predominantly activated by Gram-positive bacteria and fungi, while the Imd pathway is primarily activated by Gram-negative bacteria. Recent work has suggested that this differential activation is achieved through peptidoglycan recognition protein (PGRP)-mediated recognition of specific forms of peptidoglycan (PG). In this study, we have further analyzed the specific PG molecular requirements for Imd activation through the pattern recognition receptor PGRP-LC in both cultured cell line and in flies. We found that two signatures of Gram-negative PG, the presence of diaminopimelic acid in the peptide bridge and a 1,6-anhydro form of N-acetylmuramic acid in the glycan chain, allow discrimination between Gram-negative and Gram-positive bacteria. Our results also point to a role for PG oligomerization in Imd activation, and we demonstrate that elements of both the sugar backbone and the peptide bridge of PG are required for optimum recognition. Altogether, these results indicate multiple requirements for efficient PG-mediated activation of the Imd pathway and demonstrate that PG is a complex immune elicitor.     

PIMS modulates immune tolerance by negatively regulating Drosophila innate immune signaling

Metazoans tolerate commensal-gut microbiota by suppressing immune activation while maintaining the ability to launch rapid and balanced immune reactions to pathogenic bacteria. Little is known about the mechanisms underlying the establishment of this threshold. We report that a recently identified Drosophila immune regulator, which we call PGRP-LC-interacting inhibitor of Imd signaling (PIMS), is required to suppress the Imd innate immune signaling pathway in response to commensal bacteria. pims expression is Imd (immune deficiency) dependent, and its basal expression relies on the presence of commensal flora. In the absence of PIMS, resident bacteria trigger constitutive expression of antimicrobial peptide genes (AMPs). Moreover, pims mutants hyperactivate AMPs upon infection with Gram-negative bacteria. PIMS interacts with the peptidoglycan recognition protein (PGRP-LC), causing its depletion from the plasma membrane and shutdown of Imd signaling. Therefore, PIMS is required to establish immune tolerance to commensal bacteria and to maintain a balanced Imd response following exposure to bacterial infections.     

In vivo RNA interference analysis reveals an unexpected role for GNBP1 in the defense against Gram-positive bacterial infection in Drosophila adults

The Drosophila immune system discriminates between different classes of infectious microbes and responds with pathogen-specific defense reactions via the selective activation of the Toll and the immune deficiency (Imd) signaling pathways. The Toll pathway mediates most defenses against Gram-positive bacteria and fungi, whereas the Imd pathway is required to resist Gram-negative bacterial infection. Microbial recognition is achieved through peptidoglycan recognition proteins (PGRPs); Gram-positive bacteria activate the Toll pathway through a circulating PGRP (PGRP-SA), and Gram-negative bacteria activate the Imd pathway via PGRP-LC, a putative transmembrane receptor, and PGRP-LE. Gram-negative binding proteins (GNBPs) were originally identified in Bombyx mori for their capacity to bind various microbial compounds. Three GNBPs and two related proteins are encoded in the Drosophila genome, but their function is not known. Using inducible expression of GNBP1 double-stranded RNA, we now demonstrate that GNBP1 is required for Toll activation in response to Gram-positive bacterial infection; GNBP1 double-stranded RNA expression renders flies susceptible to Gram-positive bacterial infection and reduces the induction of the antifungal peptide encoding gene Drosomycin after infection by Gram-positive bacteria but not after fungal infection. This phenotype induced by GNBP1 inactivation is identical to a loss-of-function mutation in PGRP-SA, and our genetic studies suggest that GNBP1 acts upstream of the Toll ligand Sp?tzle. Altogether, our results demonstrate that the detection of Gram-positive bacteria in Drosophila requires two putative pattern recognition receptors, PGRP-SA and GNBP1.     

Tissue- and Ligand-Specific Sensing of Gram-Negative Infection in Drosophila by PGRP-LC Isoforms and PGRP-LE

The Drosophila antimicrobial response is one of the best characterized systems of pattern recognition receptor-mediated defense in metazoans. Drosophila senses Gram-negative bacteria via two peptidoglycan recognition proteins (PGRPs), membrane-bound PGRP-LC and secreted/cytosolic PGRP-LE, which relay diaminopimelic acid (DAP)-type peptidoglycan sensing to the Imd signaling pathway. In the case of PGRP-LC, differential splicing of PGRP domain-encoding exons to a common intracellular domain-encoding exon generates three receptor isoforms, which differ in their peptidoglycan binding specificities. In this study, we used Phi31-mediated recombineering to generate fly lines expressing specific isoforms of PGRP-LC and assessed the tissue-specific roles of PGRP-LC isoforms and PGRP-LE in the antibacterial response. Our in vivo studies demonstrate the key role of PGRP-LCx in sensing DAP-type peptidoglycan-containing Gram-negative bacteria or Gram-positive bacilli during systemic infection. We also highlight the contribution of PGRP-LCa/x heterodimers to the systemic immune response to Gram-negative bacteria through sensing of tracheal cytotoxin (TCT), whereas PGRP-LCy may have a minor role in antagonizing the immune response. Our results reveal that both PGRP-LC and PGRP-LE contribute to the intestinal immune response, with a predominant role of cytosolic PGRP-LE in the midgut, the central section of endodermal origin where PGRP-LE is enriched. Our in vivo model also definitively establishes TCT as the long-distance elicitor of systemic immune responses to intestinal bacteria observed in a loss-of-tolerance model. In conclusion, our study delineates how a combination of extracellular sensing by PGRP-LC isoforms and intracellular sensing through PGRP-LE provides sophisticated mechanisms to detect and differentiate between infections by different DAP-type bacteria in Drosophila.     

Inhibitor of apoptosis 2 and TAK1-binding protein are components of the Drosophila Imd pathway

The Imd signaling cascade, similar to the mammalian TNF-receptor pathway, controls antimicrobial peptide expression in Drosophila. We performed a large-scale RNAi screen to identify novel components of the Imd pathway in Drosophila S2 cells. In all, 6713 dsRNAs from an S2 cell-derived cDNA library were analyzed for their effect on Attacin promoter activity in response to Escherichia coli. We identified seven gene products required for the Attacin response in vitro, including two novel Imd pathway components: inhibitor of apoptosis 2 (Iap2) and transforming growth factor-activated kinase 1 (TAK1)-binding protein (TAB). Iap2 is required for antimicrobial peptide response also by the fat body in vivo. Both these factors function downstream of Imd. Neither TAB nor Iap2 is required for Relish cleavage, but may be involved in Relish nuclear localization in vitro, suggesting a novel mode of regulation of the Imd pathway. Our results show that an RNAi-based approach is suitable to identify genes in conserved signaling cascades.     

A splice variant of PGRP-LC required for expression of antimicrobial peptides in Anopheles gambiae

Members of the peptidoglycan recognition protein （PGRP） family play essential roles in different manifestations of immune responses in insects. PGRP-LC, one of seven members of this family in the malaria vector Anopheles gambiae produced several spliced variants. Here we show that PGRP-LC, and not other members of the PGRP family nor the six members of the Gram-negative binding protein families, is required for the expression of antimicrobial peptide genes （such as CEC1 and GAM1） under the control of the Imd-Rel2 pathway in an A. gambiae cell line, 4a3A. PGRP-LC produces many splice variants that can be classified into three sub-groups （LC1, LC2 and LC3）, based on the carboxyl terminal sequences. RNA interference against one LC1 sub-group resulted in dramatic reduction of CEC1 and GAM1. Over-expression of LCla and to a lesser extent LC3a （a member of the LC1 and LC3 sub-group, respectively） in the 4a3A cell line enhances the expression of CEC1 and GAM1. These results demonstrate that the LC1-subgroup splice variants are essential for the expression of CEC1 and GAM1 in A. gambiae cell line.     

Drosophila immunity: analysis of PGRP-SB1 expression, enzymatic activity and function

Peptidoglycan is an essential and specific component of the bacterial cell wall and therefore is an ideal recognition signature for the immune system. Peptidoglycan recognition proteins (PGRPs) are conserved from insects to mammals and able to bind PGN (non-catalytic PGRPs) and, in some cases, to efficiently degrade it (catalytic PGRPs). In Drosophila, several non-catalytic PGRPs function as selective peptidoglycan receptors upstream of the Toll and Imd pathways, the two major signalling cascades regulating the systemic production of antimicrobial peptides. Recognition PGRPs specifically activate the Toll pathway in response to Lys-type peptidoglycan found in most Gram-positive bacteria and the Imd pathway in response to DAP-type peptidoglycan encountered in Gram-positive bacilli-type bacteria and in Gram-negative bacteria. Catalytic PGRPs on the other hand can potentially reduce the level of immune activation by scavenging peptidoglycan. In accordance with this, PGRP-LB and PGRP-SC1A/B/2 have been shown to act as negative regulators of the Imd pathway. In this study, we report a biochemical and genetic analysis of PGRP-SB1, a catalytic PGRP. Our data show that PGRP-SB1 is abundantly secreted into the hemolymph following Imd pathway activation in the fat body, and exhibits an enzymatic activity towards DAP-type polymeric peptidoglycan. We have generated a PGRP-SB1/2 null mutant by homologous recombination, but its thorough phenotypic analysis did not reveal any immune function, suggesting a subtle role or redundancy of PGRP-SB1/2 with other molecules. Possible immune functions of PGRP-SB1 are discussed.