四种杀虫剂对两种书虱羧酸酯酶和乙酰胆碱酯酶的抑制作用

选用有机磷类杀虫剂(敌敌畏、毒死蜱、对氧磷)和氨基甲酸酯类杀虫剂(丁硫克百威),通过生物测定(药膜法)和生化测定(比色法)比较了嗜卷书虱和嗜虫书虱对所选药剂的敏感差异性。根据LC50可知嗜虫书虱对所选药剂比嗜卷书虱敏感。离体酶活性分析结果显示嗜卷书虱和嗜虫书虱的羧酸酯酶只对敌敌畏敏感,且嗜卷书虱比嗜虫书虱更敏感;4种药剂对乙酰胆碱酯酶均有强烈的抑制作用,同样是嗜卷书虱比嗜虫书虱敏感。乙酰胆碱酯酶的动力学研究结果和离体酶活性测定相一致。聚丙烯酰胺凝胶电泳分析显示,4种杀虫剂离体条件下对2种书虱的酯酶同工酶的抑制能力有明显差异,其中敌敌畏的抑制力最强;但对不同同功酶的抑制趋势(对大分子的抑制似乎较强)是一致的。酶的敏感性分析结果与生测结果比较表明,2种书虱的耐药力差异与其乙酰胆碱酯酶和酯酶对药剂的敏感性无关。如要弄清耐药力机制,需做进一步研究。     

江苏Q型烟粉虱抗性监测及其靶标基因克隆

应用成虫浸叶生测法研究了江苏省扬州、无锡和东台3个地区Q型烟粉虱Bemisia tabaci(Gennadius)田间种群对5种杀虫剂的抗药性。结果表明,与相对敏感种群相比,Q型烟粉虱田间种群已对氯氰菊酯和吡虫啉产生了低到中等水平抗性,对阿维菌素仍然表现敏感。进一步通过RCR扩增获得了长度分别为287bp和184bp的烟粉虱乙酰胆碱酯酶ace1和para-同源钠离子通道基因片断。序列分析表明,江苏Q型烟粉虱存在与有机磷抗性相关的乙酰胆碱酯酶F331W突变和与拟除虫菊酯抗性相关的钠离子通道L925I和T929V突变。     

棉蚜2个乙酰胆碱酯酶cDNA片段的基因克隆与序列分析

采用RT－PCR技术,利用简并引物从棉蚜（Aphis gossypii Glover)中克隆出2个乙酰胆碱酯酶基因的cDNA片段,Ag,ace 1l和Ag.ace2.Ag.ace1基因的cDNA片段为282bp,编码94个氨基酸;Ag.ace2基因的cDNA片段为264bp,编码88个氨基酸。扩增获得的2个乙酰胆碱酯酶基因cDNA片段所编码的氨基酸序列均与其他昆虫的乙酰胆碱酯酶基因有很高的同源性。首次从一种昆虫中克隆出2个乙酰胆碱酯酶基因片段,为同一种昆虫中存在多个乙酰胆碱酯酶基因的假设提供了直接的分子生物学证据。     

羧酸酯酶和乙酰胆碱酯酶在褐飞虱对马拉硫磷抗性发展中的作用

对室内筛选褐飞虱Nilaparvata lugens Stal对马拉硫磷抗性及羧酸酯酶、乙酰胆碱酯酶的连续变化进行了研究。结果表明,抗性发展在不同世代之间存在一定的变化。LD₅₀的最大变化发生在第3代和第5代之间。在筛选的前5代,羧酸酯酶活性上升与马拉硫磷抗性变化存在很好的相关性,而乙酰胆碱酯酶敏感性在第6代和第8代间的变化与抗性发展存在很好的相关性。可见,羧酸酯酶活性的上升在抗性发展的早期阶段起重要作用,而乙酰胆碱酯酶不敏感在抗性发展的后期阶段起更重要作用。     

AChE在水稻抗性诱导的褐飞虱凋亡中肠细胞中的定位及表达

【目的】褐飞虱Nilaparvata lugens是水稻的重要害虫之一。本研究旨在了解水稻品种抗性诱导的褐飞虱中肠细胞凋亡与细胞中乙酰胆碱酯酶(acetylcholinesterase,ACh E)的关系。【方法】从4龄Ⅰ型褐飞虱若虫中肠组织分离原代细胞,用不同抗性的水稻幼苗汁液处理第一代继代细胞,利用TUNEL染色法检测细胞的凋亡情况,再分别利用免疫组织化学和荧光定量PCR技术对ACh E进行亚细胞定位和检测其表达水平的变化。【结果】在对照组(未处理)细胞中,检测不到代表凋亡细胞核的绿色荧光,而免疫组化后阳性反应颜色很浅,表明细胞中存在ACh E的本底水平表达。感虫水稻品种TN1幼苗汁液处理细胞中,细胞的凋亡率为8%,抗性水稻B5幼苗汁液处理的细胞中有65%的细胞发生凋亡;而抗性水稻TKM-6幼苗汁液处理的细胞中则有85%的细胞发生凋亡。免疫组化的检测结果表明,所有凋亡的细胞中都存在ACh E的积累,且主要分布在细胞质中;ACh E在凋亡后期并不迁入凋亡小体中,而是集中在细胞核附近。荧光定量PCR检测表明,TKM-6,B5和TN1幼苗汁液处理的细胞中ACh E的表达量分别为对照细胞的29.9,18.4和8倍,该结果与细胞水平上免疫组化的检测结果一致。【结论】本研究结果证实了水稻品种抗性与其诱导的中肠细胞凋亡率呈正相关,且凋亡细胞的细胞质中存在ACh E的积累。这些发现为揭示ACh E在褐飞虱与水稻的互作中的功能、促进抗性水稻新品种的选育及开发新的褐飞虱防治措施提供了一定的参考信息。     

硅藻土及其混配剂对书虱的防治效果

采用拌粮法 ,用硅藻土单剂、配方 1 (硅藻土 +95 %马拉硫磷EC)、配方 2 (硅藻土 +2 . 5 %溴氰菊酯EC)、配方 3 (硅藻土 +80 %敌敌畏EC)、配方 4(硅藻土 +0 . 4%天惠虫清EC)、配方 5 (硅藻土 +5 %双氧威WP)、配方 6(硅藻土 +5 %抑太保EC)对嗜卷书虱Liposcelisbostrychophila进行防治研究 ,结果表明 :配方 4为优选配方 ,处理 2 4h后 ,3种浓度的致死率均达到 1 0 0 % ;配方 1、配方 2、配方 3、配方 5、配方 6对嗜卷书虱的致死率处理 48h后达到 1 0 0 % ;单用硅藻土处理 96h后达到 1 0 0 % ,各配方处理间差异极显著。     

褐飞虱乙酰胆碱酯酶基因全长cDNA的克隆及序列分析

利用反转录聚合酶链式反应(RT_PCR)结合快速扩增cDNA末端(RACE)技术克隆了褐飞虱Nilaparvata lugens 乙酰胆碱酯酶基因cDNA。该cDNA全长2 467 bp,包含一个1 938 bp的开放阅读框(GenBank登录号AJ852420); 在推导出的646个氨基酸残基的前体蛋白中, N端的前30个氨基酸残基为信号肽,随后的616个氨基酸残基是成熟的乙酰胆碱酯酶序列,其预测的分子量为69 418 D。在一级结构中,形成催化活性中心的3个氨基酸残基(Ser242,Glu371和His485),以及在亚基内形成二硫键的6个半胱氨酸完全保守; 位于催化功能域的14个芳香族氨基酸中有10 个完全保守。该酶的氨基酸序列与黑尾叶蝉的同源性最高,达83%。对来自23种昆虫（包括褐飞虱）的30个乙酰胆碱酯酶的聚类分析显示,褐飞虱的乙酰胆碱酯酶与其中6个Ⅱ型乙酰胆碱酯酶（AChE2）同属一个支系; 此外,只存在于昆虫AChE2中的超变区及特异的氨基酸残基,也存在于褐飞虱的乙酰胆碱酯酶中。以上结果表明,所克隆的褐飞虱的乙酰胆碱酯酶基因是一个与黑腹果蝇的orthologous型基因同源的AChE2基因。     

中国常见仓储书虱PCR-RFLP快速识别

应用PCR-RFLP技术,对我国4种常见仓储书虱即嗜虫书虱Liposcelis entomophila(Enderlein)、嗜卷书虱L.bostrychophila(Badonnel)、无色书虱L.decolor(Pearman)和小眼书虱L.paeta(Pearman)开展快速识别方法的研究。采用CTAB法从上述4种书虱体内提取基因组DNA,选用1对引物扩增16S rDNA基因区域,分别用5种限制性内切酶对PCR产物进行酶切。结果表明,PCR扩增片段大小约为500bp,用限制性内切酶DraI酶切得到的片段可将供试书虱区分开。该方法不受4种供试书虱地理种群、虫态(成虫、若虫)和性别的影响,可用于4种书虱的快速识别。     

两种书虱微卫星富集文库的构建及比较

利用链霉亲和素与生物素之间的强亲和性原理,将链霉亲和素偶联的磁珠与微卫星探针(AC)12、(TC)12、(ATC)8、(ATG)8、(AAC)8、(ATAC)6及(GATA)6退火结合后,再亲和捕捉含接头和微卫星序列的单链书虱基因组DNA限制性酶切目的片段,经PCR扩增形成双链后进行克隆、建库。结果表明本研究成功构建了嗜卷书虱和嗜虫书虱共13个微卫星富集文库,包括6个嗜卷书虱文库,7个嗜虫书虱文库,其平均阳性克隆率为71.17%。经检测发现共得到两种书虱260个微卫星位点。这两种书虱微卫星富集文库的建立和高多态性微卫星位点的筛选将为嗜卷书虱和嗜虫书虱的种群遗传与进化、基因连锁图谱构建、分子系统发育研究等提供大量分子遗传标记,对其在实仓中的持续控制提供遗传学信息。     

嗜卷书虱Cu/Zn-SOD1,Cu/Zn-SOD2和Fe/Mn-SOD的克隆及对高低温胁迫的响应

【目的】揭示嗜卷书虱Liposcelis bostrychophila超氧化物歧化酶基因在响应高低温胁迫中的作用。【方法】通过RT-PCR克隆嗜卷书虱3个超氧化物歧化酶基因Cu/Zn-SOD1,Cu/Zn-SOD2和Fe/Mn-SOD cDNA,利用生物信息学方法分析其序列特征;采用RT-qPCR技术检测Cu/Zn-SOD1,Cu/Zn-SOD2和Fe/Mn-SOD在高温(42℃)和低温(4℃)胁迫下0, 1和2 h时成虫中的相对表达量。【结果】克隆获得嗜卷书虱LbCu/Zn-SOD1,LbCu/Zn-SOD2和LbFe/Mn-SOD(GenBank登录号分别为OQ938782, OQ938783和OQ938784),开放阅读框(ORF)分别长465, 630和636 bp,分别编码154, 209和211个氨基酸,编码蛋白质相对分子量分别为15.85, 22.33和23.72 kD,等电点分别为6.17, 7.68和6.79;LbCu/Zn-SOD1和LbCu/Zn-SOD2分别具有1个和2个Cu/Zn超氧化物歧化酶特征,LbFe/Mn-SOD具有1个Fe/Mn超氧化物歧化酶特征。系...     

The vitronectin gene in rainbow trout: cloning, expression and phylogenetic analysis

Vitronectin is a major cell adhesion glycoprotein that is found in plasma and the extracellular matrix. Vitronectin consists of an N-terminal somatomedin B domain and two hemopexin-like domains and controls functions including cell adhesion, migration, haemostasis and immune defence. In order to study the molecular evolution of the complement lytic pathway regulation, we have cloned and characterized the vitronectin gene from rainbow trout (Oncorhynchus mykiss). The deduced amino acid sequence of trout vitronectin exhibits 45%, 46%, 47% and 63% identity with human, chicken, Xenopus and zebrafish orthologs, respectively. The domain architecture of the trout vitronectin, consisting of a somatomedin B domain and two hemopexin-like domains, resembles that of mammalian vitronectins. Analysis of partial genomic clones shows that trout vitronectin gene exhibits the same exon-intron organization profile as the human ortholog gene. The trout vitronectin gene is probably present as a single copy in the trout genome, showing a differential expression pattern among tissues investigated.     

Molecular cloning of, and phylogenetic analysis of, an actin in Naegleria fowleri

Abstract Two strains of Lactobacillus acidophilus Group A1, the neotype ATCC 4356 and a human isolate NCFM-N2, widely used as a dietary adjunct in milk and cultured dairy products, were transformed with plasmid DNA by electroporation. The transformation characteristics exhibited by the two L. acidophilus strains were found to differ markedly even though they appeared similar at the genomic level based on the DNA patterns of Sma I restriction fragments. To our knowledge, this is the first report of a consistent, reproducible transformation system of Lactobacillus acidophilus strains comprising the A1 DNA homology group.     

Molecular cloning and phylogenetic analysis of cereal type II metacaspase cDNA from wheat

A new cereal type II metacaspase full-length cDNA from wheat (Triticum aestivum L.) leaves, TaeMCAII, was for the first time successfully amplified and sequenced. The full-length sequence of the TaeMCAII cDNA of 1 551 bp contains a 1 218 bp open reading frame. The deduced protein encoded by the TaeMCAII cDNA consists of 405 amino acids with a calculated molecular mass of 44 kDa and an isoelectric point of 5.29. In response to wounding or heat shock, a similar sequence of ultrastructural events including the tonoplast rupture, chromatin condensation, degradation of chloroplasts and disappearance of cytoplasm and organelles were observed using transmission electron microscopy. As the observed changes in TaeMCAII mRNA level did not occur to be statistically significant wounding-induced programmed cell death (PCD) seems to be metacaspase-independent pathway. Interestingly, in PCD caused by a heat-shock treatment, the level of TaeMCAII mRNA remained unaltered until 48 h after the stress what suggests that TaeMCAII participates in later stages of PCD triggered by heat-shock. Phylogenetic analysis enabled to classify TaeMCAII as a type II metacaspase. Finally, homology modelling of the putative three-dimensional structure of the TaeMCAII protein and a topology analysis of its probable active site were performed.     

小麦种子中几丁质酶基因的扩增及序列分析

根据http://www.tigr.org中与小麦几丁质酶基因相关的序列TC187877,设计引物,分别从小麦品种Gamenya和苏麦3号中扩增到大小约为1 000bp的片段。经序列测定和软件分析比较,发现这些片段所编码的蛋白质氨基酸序列,都有CHITINASE-19.1和CHITINASE-19.2的基序,为第II类几丁质酶基因。扩增的核酸序列在GenBank上发表,登录号分别为AY973229和AY973230。     

Molecular cloning and phylogenetic analysis of the small cytoplasmic RNA from Listeria monocytogenes

A molecular cloning strategy has been designed to isolate the gene that encodes the small cytoplasmic RNA (scRNA) component of bacterial signal recognition particles. Using this strategy a putative Listeria monocytogenes scRNA lambda gt11 recombinant clone was isolated. A previously described complementation assay developed to genetically select functional homologues of 4.5S RNA and scRNA of bacteria confirmed that the lambda gt11 recombinant clone isolated encoded for the scRNA from L. monocytogenes. A secondary structure for this scRNA is proposed and a phylogenetic comparison of the 276 base L. monocytogenes scRNA with previously characterised Gram-positive bacterial scRNAs is also presented.     

Zebrafish caspase-3: molecular cloning, characterization, crystallization and phylogenetic analysis

Apoptosis plays an important role in maintaining the normal function of various tissues and organs in different species. Caspase-3 is a terminal caspases which plays an important role in the execution of apoptosis in all vertebrates. It was cloned from zebra fish embryos and its properties were identified through Western blotting and biological activity. In the cells over-expressing caspase-3, Western blotting with an anti-His-tag antibody confirmed the presence of caspase-3 in the three bands that were proposed to correspond to the precursor form (33 kDa), the mature forms processed at the prodomain alone (29 kDa, large subunit) and small sub unit (13 kD). Fish kidney cells were transiently co-transfected with the beta-galactosidase reporter gene and either vector alone (mock), pZCASP3His (caspase-3) or pZCASP3His mutant (caspase-3 mutant). After 72 h following transfection of fish kidney cells, 35% of cells transfected with the zebra fish caspase-3 construct, pZCASP3His, showed apoptotic morphology when compared with cells transfected with the mock vector or an expression construct (pZCASP3His mutant) encoding the caspase-3 mutant lacking Cys. The fusion proteins were expressed in Escherichia coli, isolated from cell lysates by nickel-affinity column chromatography, and cleaved with thrombin. A thrombin cleavage recognition site was positioned at the fusion junction to release the caspase-3 from the fusion protein. Phylogenetic analysis showed that the cloned zebra fish caspase was a member of the caspase-3 subfamily with approximately 60% identity with caspase-3 from Xenopus, chicken and mammals. We have obtained structural information by X-ray crystallography. Orthorhombic crystals of the caspase-3 that diffracted to 1.8 A were obtained in a mixture of 0.1 M imidazole (pH 6.0) and 0.4 M NaOAc (pH 7.0 -7.5), containing 30% glycerol. The space group is C222 with cell dimensions of a = 36.07 A, b = 38.80 A, c = 135.20 A.     

In vitro and in silico cloning of Xenopus laevis SOD2 cDNA and its phylogenetic analysis

By using the methodology of both wet and dry biology (i.e., RT-PCR and cycle sequencing, and biocomputational technology, respectively) and the data obtained through the Genome Projects, we have cloned Xenopus laevis SOD2 (MnSOD) cDNA and determined its nucleotide sequence. These data and the deduced protein primary structure were compared with all the other SOD2 nucleotide and amino acid sequences from eukaryotes and prokaryotes, published in public databases. The analysis was performed by using both Clustal W, a well known and widely used program for sequence analysis, and AntiClustAl, a new algorithm recently created and implemented by our group. Our results demonstrate a very high conservation of the enzyme amino acid sequence during evolution, which proves a close structure-function relationship. This is to be expected for very ancient molecules endowed with critical biological functions, performed through a specific structural organization. The nucleotide sequence conservation is less pronounced: this too was foreseeable, due to neutral mutations and to the species-specific codon usage. The data obtained by using AntiClustAl are comparable with those produced with Clustal W, which validates this algorithm as an important new tool for biocomputational analysis. Finally, it is noteworthy that evolutionary trees, drawn by using all the available data on SOD2 nucleotide sequences and amino acid and either Clustal W or AntiClustAl, are comparable to those obtained through phylogenetic analysis based on fossil records.     

Identification, molecular cloning, and phylogenetic analysis of a non-respiratory pseudo-hemocyanin of Homarus americanus

Copper-containing hemocyanins serve to transport oxygen in many arthropod species. Here I describe the identification and cDNA cloning of a structurally closely related non-respiratory pseudo-hemocyanin (PHc) of the American lobster, Homarus americanus. This protein has lost the ability to bind copper and, therefore, oxygen because a histidine residue in copper-binding site A is replaced by tyrosine. Like many arthropod hemocyanins, PHc forms a hexamer. It consists of two different subunit types of 660 and 661 amino acids, respectively, that share a 94.4% sequence identity. Whereas Homarus hemocyanin is produced in the hepatopancreas, PHc is synthesized by the ovaries and the heart tissue. Because different levels of PHc were observed in distinct individuals, I propose an association of the synthesis of this protein with the molting or reproduction cycle, similar to the hexamerins, insect storage proteins that are also related to the hemocyanins. However, phylogenetic analyses show that PHc derived independently from crustacean hemocyanins. Therefore, Homarus PHc is a member of a new class within the growing hemocyanin protein superfamily.