淡色库蚊酯酶等位基因及其在自然种群中的频率分布

酯酶基因扩增所产生的酯酶活性升高是库蚊Culex pipiens对有机磷杀虫剂抗性的主要机理之一。采用分子杂交技术和限制性酶切片段长度多态性(RFLP)分析,已鉴定出多种酯酶等位基因类型。该文通过酯酶基因特异性片段的PCR扩增及扩增片段的酶切片段分析,对淡色库蚊Culex pipiens pallens四种有机磷抗性品系的酯酶等位基因进行分型,并测定分析自然种群中不同酶型的频率分布。研究结果表明：PCR分型方法具有快速、准确的特点。不同的有机磷杀虫剂对酯酶等位基因具有明显的选择作用。双硫磷品系为B₁型;毒死蜱和敌百虫品系为B₂型;马拉硫磷品系为B₁型和B₁/B₂杂合型。不同地区采集的种群表现出不同的酶型频率分布。该文就杀虫剂对酯酶等位基因选择作用及自然种群的酶型频率分布进行了讨论。     

几种杀虫药剂敏感蚊类酯酶多态性的研究

通过蚊虫酯酶蛋白的淀粉凝胶电泳分析和基因组DNA的限制性酶切片段长度多态性(RFLPs)比较, 对尖音库蚊Culex pipiens、三带喙库蚊Culex tritaeniorhynchus和中华按蚊Anopheles sinensis有机磷杀虫药剂敏感种群的酯酶蛋白和结构基因的多态性进行分析。发现在蛋白质水平上,三带喙库蚊敏感种群(n=54)在酯酶α和β位点分别存在2个和3个等位基因,在DNA水平上有2.9%的个体具有与酯酶β1¹基因1.3 kb Cdna片段同源的1.3 kb单拷贝带存在。发现中华按蚊敏感种群 (n= 50)中具有低活性的非特异性酯酶存在,在蛋白质水平上,酯酶α和β位点各有一个等位基因;在DNA水平上,通过对单个蚊虫基因组DNA的研究未发现有与酯酶β1¹基因同源的酯酶编码基因的存在。对尖音库蚊北京敏感种群(n= 64)的研究发现,在酯酶α和β位点都存在5个等位基因,在DNA水平上,使用一个限制性内切酶(EcoRI),15只蚊虫的样本在酯酶β位点发现了5个等位基因,说明在尖音库蚊北京敏感种群的酯酶β基因周围存在着较大的中性多态性,在有机磷杀虫剂的选择下,这些中性多态性可能会成为基因扩增的潜在因素。     

有机磷抗性致倦库蚊种群中酯酶基因扩增的定量分析

致倦库蚊Culex qinquefasciatus是丝虫病的主要传染媒介。通过生物测定、单个蚊虫酯酶α2和β2基因拷贝数分析和酯酶β基因序列比较, 分析了抗性水平、抗性相关基因在种群中的分布及其基因拷贝数等的抗性分子特征。应用快速PCR仪（realtime quantitatIve PCRs）直接检测库蚊中酯酶基因和mRNA拷贝数。结果显示：上海致倦库蚊对对硫磷的抗性LC₅₀为8.12, 酯酶活性升高是上海致倦库蚊种群对有机磷杀虫药剂产生抗性的主要机理。编码致倦库蚊酯酶β的氨基酸序列同编码尖音库蚊酯酶B1的氨基酸序列相比同源性为98%;同致倦库蚊酯酶B2氨基酸序列相比同源性为100%,同环蹶库蚊酯酶B3氨基酸序列相比同源性为90%, 上海致倦库蚊中酯酶α和β基因均扩增。有机磷抗性的上海和PellRR蚊虫种群中单个蚊虫酯酶α2 和β2定量基因拷贝数均不同,其同一蚊虫个体的酯酶α2 比酯酶β2基因的拷贝数高,但没有明显的规律性,酯酶结构基因的扩增是上海致倦库蚊种群对有机磷杀虫药剂抗性的主要机理,估计在野外种群的杂合个体中存在多种调控机制。     

库蚊野生种群扩增等位基因重组现象分析

羧酸酯酶 (carboxylesterases)的过量产生是库蚊Culexpipiens对有机磷杀虫剂 (OP)产生抗性的主要机制。由est 3和est 2组成的酯酶超级基因座 (estersuper locus)的基因扩增是引起酯酶基因扩增的主要遗传学基础。通过淀粉电泳研究了采自广州、佛山、郑州的库蚊野生蚊虫种群 ,发现在这些种群中存在着扩增等位基因重组现象。该现象可能是蚊虫受到杀虫药剂的选择压力、等位基因多样性和等位基因型频率的影响。这将提供一个研究抗性进化的自然模型。     

不同地区尖音库蚊复合组抗药笥的分子特征

通过生物测定、蛋白质电泳和单个蚊虫基因组Southern杂交三种方法对三个不同地区的尖音库蚊复合组蚊虫Culex pipiens complex的抗性水平、抗性相关基因在处群中的分布及抗性分子特征进行了研究。采自山东高密、北京和云南昆明的尖音库蚊复合组蚊虫4龄幼虫对敌敌畏的抗性水平也很高（约30倍）,三个种群对仲丁威的抗性与北京第三系没有明显的区别。淀粉电泳的结果表明：高密和北京种群中存在过量产生的非特异性酯酶Estβ1^1,昆明种群中存在过量产生的非特异性酯酶Estα2和Estβ2,单个蚊虫基因组Southern杂交的结果表明：酯酶Estβ1^1、Estα2和Estβ2的结构基因发生了不同程度的扩增导致酯酶的过量产生。     

中美有机磷杀虫药剂抗性库蚊复合种群酯酶基因扩增的研究(英文)

用脉冲电场凝胶电泳技术,从北京和美国加州的有机磷杀虫药剂抗性库蚊复合品系中分离出一条49kb的酯酶基因扩增片段。该片段可能是一个完整的扩增单元,也是迄今分离出的最大的一条昆虫酯酶基因扩增片段,扩增基因结构的研究正在进行之中。本文还重点讨论了DNA大片段的制备方法和脉冲电场电泳分离技术。     

杭州地区尖音库蚊复合组的抗药性动态

用生物测定和淀粉电泳技术对采自杭州市和上海市的尖音库蚊复合组(Culex pipienscomplex)蚊虫的抗性水平及与抗性有关的酯酶进行了研究。抗性水平的测定结果表明:与S-LAB敏感系相比,杭州市种群对DDVP、对硫磷、毒死蜱、邻仲丁基苯基甲基氨基甲酸酯和残杀威的抗性分别是S-LAB敏感品系的3.9,7.6,1.6,1.6,2.5倍。利用淀粉凝胶电泳技术分析了野生种群中抗性羧酸酯酶(EST)等位酶的基因表型及其频率。4个种群中都存在着世界范围内广泛分布的过量产生的非特异性酯酶Estβ1和Estα2/β2,上海种群主要以高活性的酯酶Estβ11为主(98.3%),除了这些基因以外,2004年杭州市下城区又出现了1种新的酯酶基因(50%)。     

家蚕基因特异性CAPs标记获得及其分子系统学应用

选取家蚕attacin和alpha-amylase基因序列,设计特异性引物,在家蚕品系P50、C108和子一代 (F1) 中扩增。分别采用4种不同的限制性内切酶对扩增产物酶切,最后每个基因都获得了一个CAPs分子标记。依据所得的两个CAPs分子标记对12个品系的家蚕遗传多样性进行了初步研究,构建了其分子系统树。     

限制性内切酶介导的重叠延伸在人环氧合酶-1(COX-1)基因克隆中的应用

建立一种用于克隆全长基因的、限制性内切酶介导的重叠延伸法 .对全长基因进行分段扩增 ,并利用适当的限制性内切酶对基因序列内相应的限制性位点进行酶切 ,从而使分段扩增片段得以重叠并互为模板 ,在DNA聚合酶的作用下延伸获得全长基因 .将环氧合酶 1 (COX 1 )基因的外显子 9巧妙地拼接到了缺失外显子 9的COX 1cDNA片段中 ,获得了COX 1基因的全长cDNA .该方法分 3步进行 .首先 ,通过RT PCR分别扩增跨外显子 9的cDNA片段和缺失外显子 9的cDNA片段 ,并克隆到pMD1 8 T载体上 ;其次 ,PCR扩增外显子 9片段 ,限制性内切酶StuI酶切缺失外显子9cDNA片段的重组质粒 ,二者以一定的比例混合 ,互为模板 ,在pfuDNA聚合酶的作用下进行延伸 ,从而产生一个双链的DNA分子 .最后 ,以延伸产物为模板 ,用COX 1cDNA两端的引物进行PCR扩增 ,产生包含外显子 9的COX 1基因的全长cDNA .这种限制性内切酶介导的重叠延伸方法 ,对于克隆mRNA剪接水平上受调控的基因尤为有用 ,同时也为基因的重组和修饰提供一个新的思路     

一种基因拷贝的过量存在能产生对杀虫剂的抗性

虽然还不能马上解决昆虫对有机磷杀虫剂的抗性问题,但法国的 Claude Mouchès和他的同事发现,一种基因拷贝的过量存在能使蚊子对杀虫剂产生抗性,这为着手解决这个问题开辟了新途径。该基因是一种酯酶 B1基因。据这个法国小组的测定表明,一种能抗有机磷杀虫剂的加利福尼亚蚊子品系所含的酯酶 B1较那些对该杀虫剂敏感的品系要多500倍。他们指出,能编码酯酶的这种基因在该抗性品系中至少能增加到250倍。     

Mosquito carboxylesterases: a review of the molecular biology and biochemistry of a major insecticide resistance mechanism

The major mechanism of organophosphorus insecticide resistance in Culex mosquitoes involves the elevation of one or more esterases. The general mechanism underlying this resistance is the amplification of the structural genes. This review covers the classification of the mosquito esterases in the context of classical esterase nomenclature. The function of the amplified esterases and the structure of the amplified DNA on which they occur are also described. Implications of information on the esterase amplicons are discussed in relation to the evolution and migration of insecticide resistance in Culex.     

Identification of two distinct amplifications of the esterase B locus inCulex pipiens (L.) mosquitoes from mediterranean countries

Two new highly active esterases were detected by starch electrophoretic studies inCulex pipiens mosquitoes from the area of Montpellier (France) and from Cyprus. We demonstrate here that both the French and the Cyprus esterases B are overproduced due to amplification of the coding gene. The production of the esterase B is approximately 50- and 500-fold higher in mosquitoes from France and Cyprus, respectively, than in susceptible insects, whereas the number of gene copies is about 25 and 250. Differences of about 7- and 95-fold were also found in the degree of chlorpyrifos resistance. RFLP comparison of the amplified region containing the esterase B gene revealed large differences between French and Cyprus mosquitoes. It thus appears that two distinct haplotypes with an esterase B gene coding an enzyme with identical electrophoretic mobility have been amplified. We therefore named the haplotypes in mosquitoes from France and Cyprus B4 and B5, respectively. The estimated genetic distance between these two haplotypes is not smaller than those observed in all pair comparisons of other known esterase B haplotypes. These results are discussed in the context of amplification phenomena.This work was supported in part by Grant 89.1540 from the Ministère de l'Education Nationale and by the CNRS/Programme Environnement.     

Esterases A5-B5 in organophosphate-resistant Culex pipiens from Italy

Abstract. Culex pipiens mosquitoes from Lignano city, Udine province, northeast Italy, were found to carry over-produced non-specific esterases Al, A2-B2 and A4-B4 or A5-B5, detected by starch gel electrophoresis, giving multiple resistance to organophosphorus insecticides. In order to differentiate between A4-B4 and A5-B5 esterases, the latter known only from Cyprus whereas the former is widespread in Italy and elsewhere, restriction fragment length polymorphism (RFLP) analysis was performed at the esterase B locus. Both B4 and B5 haplotypes were found. This is the first record of A5-B5 esterase-mediated resistance in continental Europe.     

An overview of the evolution of overproduced esterases in the mosquito Culex pipiens

Insecticide resistance genes have developed in a wide variety of insects in response to heavy chemical application. Few of these examples of adaptation in response to rapid environmental change have been studied both at the population level and at the gene level. One of these is the evolution of the overproduced esterases that are involved in resistance to organophosphate insecticides in the mosquito Culex pipiens. At the gene level, two genetic mechanisms are involved in esterase overproduction, namely gene amplification and gene regulation. At the population level, the co-occurrence of the same amplified allele in distinct geographic areas is best explained by the importance of passive transportation at the worldwide scale. The long-term monitoring of a population of mosquitoes in southern France has enabled a detailed study to be made of the evolution of resistance genes on a local scale, and has shown that a resistance gene with a lower cost has replaced a former resistance allele with a higher cost.     

不同地区致倦库蚊种群相关酯酶 基因的特征分析

分别从广州、沙市、武汉近郊采集到致倦库蚊Culex pipiens quinquefasciatus,对单只蚊虫进行淀粉凝胶电泳和Southern杂交方法的分析结果表明：3个实验种群中均分布有与抗性有关的高活性酯酶β1¹,酯酶α2/β2分布于广州实验种群中;广泛分布于地中海地区尖音库蚊Culex pipiens种群中的酯酶α4/β4、α5/β5在以上3个种群中均不存在。但是,在3个实验种群中均发现存在有一对新的高活性酯酶α8/β8,其电泳迁移率和限制性酶切片段均与目前已报道的几种高活性酯酶不同。含这两对新酯酶的蚊虫将应进一步从种群中纯化,纯合蚊虫新品系做分子特征的研究。     

Molecular and kinetic evidence for allelic variants of esterase Estβ1 in the mosquito Culex quinquefasciatus

Elevated esterase Estbeta1 was purified from larvae of newly isolated strains of the mosquito Culex quinquefasciatus from Colombia (COL) and Trinidad (TRI) with resistance to organophosphate (OP) insecticides. Insecticide interactions were compared with those of elevated Estbeta1(2) from the OP-resistant Habana strain and the non-elevated Estbeta1(3) from the susceptible PelSS strain. On the basis of insecticide binding efficiency, all elevated Estbeta1 esterases were readily distinguishable. Differences between the EcoRI restriction fragment patterns of the amplified estbeta1 gene in COL and TRI strains compared with each other, and between amplified estbeta1(1), estbeta1(2) and the non-amplified estbeta1(3), suggest differences in their nucleotide sequence. Considering their variable insecticide binding efficiencies, these genetic differences would imply that, in contrast to estalpha2 and estbeta2, amplification of estbeta1 has occurred several times independently. Generally, the elevated Estbeta1s were more reactive with insecticides than the non-elevated Estbeta1(3). This supports the hypothesis that the elevated esterase-based mechanism confers resistance through amplification of alleles coding for esterases which have a greater specificity for the insecticides they sequester than the esterases coded by their non-amplified counterparts.     

Molecular comparisons of the Culex pipiens (L.) complex esterase gene amplicons

The amplification of carboxylesterase genes is a mechanism of organophosphate resistance in Culex mosquitoes. Amplified carboxylesterase genes from an insecticide resistant Culex pipiens strain collected in Cyprus were analysed and compared to other Culex amplified carboxylesterase alleles. A 12 kb section of genomic DNA containing two gene loci coding for carboxylesterase alleles A5 and B5 was cloned and sequenced. A comparison between this amplicon and one from a strain with co-amplified carboxylesterase alleles A2 and B2 revealed a number of differences. The intergenic spacer was 3.7 kb in length in the A5-B5 amplicon (2.7 kb in A2-B2) and contained putative Juan and transposable elements upstream of B5. A fragment of a gene with high homology to aldehyde oxidase was also present immediately downstream of A5. The comparison revealed no differences that would explain the successful spread of the A2-B2 amplicon worldwide whilst the A5-B5 amplicon is restricted to the Mediterranean.