【作者】 唐培安；

【导师】 王进军；

【作者基本信息】 西南大学 ， 农药学， 2009， 博士

【摘要】 书虱(psocids)属于虱啮目Psopotera、书虱科Liposcelididae、书虱属Liposcelis,是一类重要的储藏物害虫,大量发生时可造成严重的经济损失,而且对化学药剂的抗性发展很快,已经引起了全世界储藏物工作者的高度重视。在我国发生为害最为严重的种类有嗜卷书虱Liposcelis bostrychophila、嗜虫书虱L.entomophila、无色书虱L.decolor以及小眼书虱L.paeta等。本研究在国家自然科学基金(30570231)和教育部新世纪优秀人才支持计划(NCET-04-0854)的资助下,重点研究了嗜卷书虱、嗜虫书虱和无色书虱酯酶(包括乙酰胆碱酯酶和羧酸酯酶)及乙酰胆碱受体的分子生物学特性,获得了以下主要研究结果:1嗜卷书虱酯酶基因克隆及其mRNA表达水平研究1.1嗜卷书虱乙酰胆碱酯酶基因克隆及序列分析采用RT-PCR和RACE技术成功克隆获得了嗜卷书虱第2个乙酰胆碱酯酶(AChE)基因的全长序列(GenBank登录号:FJ647185),该基因全长3316bp,开放阅读框(ORF)2814 bp,编码一个937个氨基酸组成的AChE前体蛋白,其中包括了由23个氨基酸组成的信号肽,成熟蛋白的分子量为104.8 kDa,理论等电点为6.63。序列同源性分析表明,该基因是与黑腹果蝇旁系同源的Ⅰ型AChE的基因,因此将其命名为Lb acel,它所编码的蛋白质为ACHE1。嗜卷书虱2个AChE基因之间的同源性较低,仅为38.75%,但它们与各自同类型的其它昆虫的AChE具有很高的同源性。序列比对分析发现,嗜卷书虱AChE1具有AChE家族所有的保守性功能位点,如:催化三联体、氧阴离子洞等。另外,应用蛋白结构同源建模工具SWISS-MODEL,以人丁酰胆碱酯酶(1p0i:A)的蛋白晶体结构为模型,对嗜卷书虱AChE1的三维结构进行了模拟,并从三维结构中发现了嗜卷书虱AChE1的酶解活性位点。1.2嗜卷书虱乙酰胆碱酯酶基因mRNA表达水平研究利用Real Time PCR技术对嗜卷书虱2个AChE基因在不同品系、不同发育阶段以及药剂处理前后的mRNA表达水平进行了研究。结果表明,抗性品系(敌敌畏抗性品系和磷化氢抗性品系)2个AChE基因的表达水平均显著高于敏感品系(P＜0.05)。使用敌敌畏或磷化氢熏蒸诱导后,嗜卷书虱体内2个AChE基因的表达水平均显著升高(P＜0.05)。其中嗜卷书虱2个AChE基因均在2龄若虫期表达水平最高,而在1龄若虫和成虫期的表达水平最低。1.3嗜卷书虱羧酸酯酶基因克隆及序列分析利用RT-PCR和RACE技术成功克隆获得嗜卷书虱2个羧酸酯酶(CarE)基因的全长序列,分别命名为Lb est1(GenBank登录号:EU854151)和Lb est2(GenBank登录号:EU854152)。其中Lbest1基因全长为2049 bp,ORF 1713 bp,编码570个氨基酸组成的前体蛋白,N端19个氨基酸为信号肽;Lb est2基因全长为2525 bp,编码617个氨基酸组成的前体蛋白,其中包括了由17个氨基酸组成的信号肽。根据在线软件ScanProsite的算法原理,从2个基因的氨基酸序列中均找到了CarE的2个保守结构域:丝氨酸活性中心(Lb est1:FGGDPNKVTIFGESAG;Lb est2:FGGDPNRITLFGESAG)和保守的二硫键形成位点(Lb est1:EDCLFLNVFTP;Lb est2:EDCLYLNIYSP)。序列同源性分析表明,嗜卷书虱2个CarE基因与其它昆虫酯酶基因之间的同源性较低,但在活性中心处的序列则高度保守。1.4嗜卷书虱羧酸酯酶基因mRNA表达水平研究嗜卷书虱CarE基在定量分析结果表明,Lb est2基在在敌敌畏和磷化氢抗性品系中的表达量分别为敏感品系的1.91和1.42倍,且差异达显著水平(P＜0.05)。药剂处理也可诱导CarE基因表达量的显著升高(P＜0.05)。Lb est1在抗性和敏感品系中的表达水平差异虽不显著(P＞0.05),但经敌敌畏和磷化氢处理后其表达水平分别升高了1.76和1.47倍。CarE基因在不同发育阶段mRNA表达量的分析结果表明,Lb est1的表达量随着该虫生长发育的进行逐渐降低,至成虫期时表达水平最低;相反,L6 est2在若虫期表达水平较低,而在成虫期表达水平最高。2嗜虫书虱乙酰胆碱酯酶基因克隆及其mRNA表达水平研究2.1嗜虫书虱乙酰胆碱酯酶基因克隆及序列分析利用RT-PCR和RACE技术成功克隆获得嗜虫书虱2个AChE基在的全长序列,分别命名为Le ace1(GenBank登录号:EU854149)和Le ace2(GenBank登录号:EU854150)。其中Le ace1基因全长1958 bp,ORF 1890 bp,编码629个氨基酸组成的蛋白质;Le ace2基因全长为2171 bp,ORF 1914 bp,编码637 aa的前体蛋白,N端20个氨基酸为信号肽,成熟蛋白的分子量为72.2 kDa,理论等电点为4.99。这2个AChE氨基酸序列之间的同源性较低(35.73%)。通过与黑腹果蝇和加州电鳐AChE的序列比对发现,嗜虫书虱2个AChE基因均具有AChE家族的所有保守性功能位点,如:催化三联体、氧阴离子洞等。利用蛋白质结构同源建模工具,分别以人丁酰胆碱酯酶(1p0i:A)和果蝇乙酰胆碱酯酶(1d×4:A)的蛋白晶体结构为模板,对嗜虫书虱2个AChE的三维结构进行同源建模,并在三维结构中发现了AChE的酶解活性位点,证明嗜虫书虱体内也存在2个AChE基因。2.2嗜虫书虱β-actin基因克隆及乙酰胆碱酯酶基因mRNA表达水平研究目前关于嗜虫书虱的分子生物学研究较少,在GenBank中没有可用作内参基因的序列,因此本研究从嗜虫书虱体内克隆获得β—actin基因片段(GenBank登录号:FJ041117),该片段长度为822 bp,编码273个氨基酸残基,同源性比对分析表明该片段与其它昆虫的β—actin基因具有很高的同源性。以嗜虫书虱β-actin作为内参基因,通过Real Time PCR技术研究发现,嗜虫书虱Le ace1基因mRNA的表达量是Le ace2基因的1.6倍,并且经涕灭威和马拉硫磷处理后,该虫体内的2个AChE基因的表达量均显著升高(P＜0.05)。3无色书虱乙酰胆碱酯酶基因克隆及其系统进化分析利用RT-PCR和RACE技术克隆获得了无色书虱AChE1基因(Ld ace1)的3’端序列(GenBank登录号:FJ647186)和ACHE2基在(Ld ace2)的全长序列(GenBank登录号:FJ647187)。其中Ld ace2基因全长为2111 bp,ORF 1917 bp,编码638个氨基酸组成的前体蛋白,成熟蛋白的分子量为71.9 kDa,理论等电点为4.70。经序列比对找到了AChE家族的所有保守性功能位点,并以黑腹果蝇AChE(1d×4:A)的蛋白晶体结构为模板进行蛋白结构同源建模。Ld ace1基因仅获得了3’端1616 bp的序列,该序列编码500个氨基酸残基,且与嗜卷书虱和嗜虫书虱的AChE1同源性高达91%和90%;经序列比对分析发现,除了缺少形成一对二硫键的半胱氨酸外,无色书虱AChE1包含了AChE家族的所有保守性功能位点。通过从GenBank中收集到的多个物种的AChE氨基酸序列,结合嗜卷书虱、嗜虫书虱和无色书虱的AChE氨基酸序列,利用MEGA4.1构建了AChE的分子进化树,从生成的分子进化树可以看出,所有的AChE大体上可以分为4个大的类群:昆虫Ⅰ型AChE类群、脊椎动物AChE类群、昆虫Ⅱ型AChE类群利线虫AChE类群。本研究中的3种书虱AChE分别被划归为昆虫Ⅰ型AChE类群和昆虫Ⅱ型AChE类群,且具有最近的进化关系。昆虫的两类AChE严格的区分,其中Ⅰ型AChE先与脊椎动物AChE聚合后才与Ⅱ型AChE相聚,说明两类AChE基因在物种分化前就已经分化。4嗜卷书虱烟碱型乙酰胆碱受体基因克隆及其mRNA表达水平研究利用RT-PCR和RACE技术成功克隆获得了嗜卷书虱2个烟碱型乙酰胆碱受体(nAChR)亚基的全长序列,分别命名为Lb a1(GenBank登录号:EU871527)和Lb a8(GenBank登录号:EU871526)。其中L6 a1基因全长为2025 bp,ORF 1644 bp,编码一个547个氨基酸组成的前体蛋白,N端18个氨基酸为信号肽。Lb a8基因全长为1763 bp,ORF 1608 bp,编码535个氨基酸组成的AChE前体蛋白,包括了由22个氨基酸组成的信号肽。同源性比对分析发现,这2个亚基与其它昆虫的nAChR亚基具有很高的同源性,尤其与头虱的a亚基同源性高达90%以上。序列比对结果农明,嗜卷书虱nAChR 2个亚基均具有长的N端胞外区和4个跨膜区等保守结构,其中在N端胞外区包含1个Cys-loop以及6个乙酰胆碱的结合位点(loops A-F)。另外,这2个亚基都含有a型亚基所特有的2个相连的半胱氨酸残基。系统进化分析结果显示,这2个亚基分别属丁a1类和a8类亚基。根据在线软件预测,Lb a1亚基含有15个磷酸化位点和2个糖基化位点,Lb a8基含有9个磷酸化位点和1个糖基化位点。利用Real Time PCR技术研究了这2个亚基mRNA的表达水平。结果表明,Lb a1亚基在成虫期的表达水平显著高于若虫期(P＜0.05),而在若虫期以1龄若虫期表达量最高,3龄若虫期的表达水平最低;Lb a8亚基在3龄和4龄若虫期表达量最低,而在1龄若虫和成虫期的表达水平最高。Lb a1在1龄、2龄、3龄、4龄若虫及成虫中的表达水平分别是Lb a8的2.03、2.55、4.77、6.54、和5.28倍,且它们之间的差异均达到显著水平(P＜0.05)。5温度对无色书虱发育和繁殖的影响通过比较无色书虱在不同温度条件下的发育速率、存活率、内禀增长率等生命参数以明确无色书虱在不同温度条件下的发生发展规律。研究发现,无色书虱若虫期雌性4龄,而雄虫3龄。温度对无色书虱雌虫和雄虫的卵期、若虫期及整个未成熟期的发育历期均有极显著的影响(P＜0.01)。在供试温度(20-37.5℃)范围内,无色书虱雌虫的发育历期从20℃的46.2 d(雄虫为41.8 d)逐渐降低到35℃的16.1 d(雄虫为13.6 d),但在37.5℃极端高温下无色书虱的发育历期有所延长(雌虫为20.0 d,雄虫为16.3 d)。无色书虱未成熟期存活率在32.5℃条件下最高(57.3%),而在37.5℃下最低(19.0%)。无色书虱的平均每雌产卵量在32.5℃下最大(130.4粒),而在37.5℃下最小(24.7粒)。在供试的8个温度条件下,无色书虱实验种群在32.5℃时内禀增长率(r_m)和净增值率(R_o)最大(分别为0.0609,16.61)、种群翻倍时间最短(11.39 d)。使用Weibull方程拟合无色书虱种群的存活曲线,在20-32.5℃下种群的l_x曲线属DeeveyⅠ型曲线,即绝大多数个体能实现其平均寿命;而在其它温度条件下属于DeeveyⅢ型曲线,即无色书虱卵及低龄若虫期的死亡率较高。总体来看,本学位论文较为系统的研究了书虱体内酯酶(包括乙酰胆碱酯酶和羧酸酯酶)以及乙酰胆碱受体的分子生物学特性,这为今后深入研究书虱抗药性分子机理奠定了坚实的基础。特别是所研究的3种书虱体内均存在2个乙酰胆碱酯酶基因的结果,为昆虫体内具有2个不同的AChE基因提供了又一直接证据。研究的内容和结果,不仅在理论上深化了对书虱分子毒理学的研究,充实生物进化及遗传变异的研究内容,在实践上可以为开发害虫抗性分子检测技术、制订延缓抗性发展和抗性治理方案等提供了重要的理论指导。更多还原

【Abstract】 Psocids,belonging to Liposcelididae,Psocoptera,are worldwide and commonly found in various processed and unprocessed dry foods.Outbreaks of psocids could pose an alarming threat to stored product.Routine fumigations of warehouses and storage facilities with methyl bromide and PH₃ have failed to control these pests.In addition,the rapid development of resistance to chemical insecticide by psocids has also been reported.In China,the commonly occurred psocid species are Liposcelis bostrychophila,L.entomophila,L.decolor,and L.paeta.In this dissertation,the molecular characteristics of acetylcholinesterases（ACHE）,carboxylesterase（CarE）,and nicotinic acetylcholine receptor（nAChR） in L.bostrychophila,L.entomophila,and L.decolor were investigated.The project is supported by National Natural Science Foundation of China（30570231） and the Program for New Century Excellent Talents in University（NCET-04-0854）.The major results are summarized as follows:1 Gene cloning of esterases and mRNA expression level in L. bostrychophila1.1 Gene cloning and sequence analysis of AChE in L.bostrychophilaThe second full length cDNA encoding AChE（GenBank Accession No.:FJ647185） was cloned from L.bosrtychophila by the methods of reverse transcriptase PCR（RT-PCR） and rapid amplification of cDNA ends（RACE）.The complete cDNA of this gene consists of 3316 bp with an open reading frame（ORF） of 2814 bp,encoding a protein of 937 amino acids residues with the putative signal peptide of 23 residues.The mature protein has a molecular weight of 104.8 kDa with an isoelectric point of 6.63.This gene is paralogous to AChE of Drosophila melanogaster by sequence homology analysis,and thus named Lb acel.Although a low identity between two AChEs of L.bostrychophila（38.75%）,both of them share high identity to the same type of AChEs from other insect species.ACHE1 from L.bostrychophila possesses 92%and 90%amino acid reside identities to those of L.entomophila and L.decolor.Also,the identities to ACHE1 from Spodoptera exigua,Nephotettix cincticeps and Helicoverpa assulta are higher than 70%.According to sequence alignment,the predicted amino acids contained all typical residues of AChE such as the catalytic triad and the oxy-anion hole.Homology modeling of 3-D structure of ACHE1 from L. bostrychophila is constructed using Homo sapiens（1p0i:A） native BuChE structure as template by SWISS-MODEL.The catalytic triad were found and denoted in the 3-D structure of AChE1 from L.bostrychophila referring to Torpedo californica. 1.2 mRNA expression levels of two AChE genes from L.bostrychophilaThe mRNA expression levels of two ace genes from L.bostrychophila in different strains, development stages,and insecticide treatments were studied using Real Time PCR.The results showed that the expression levels of two ace genes in resistant strains（DDVP_R and PH_3R） were significantly higher than those of susceptible strain（P＜0.05）.After treated by dichlorvos（DDVP） or phosphine（PH₃）,the expression levels of two ace genes were all significantly increased than control.The highest expression level of two ace genes was detected at the second stadium and the lowest was at the first stadium and adult stage.1.3 Gene cloning and sequence analysis of CarE in L.bostrychophilaTwo full length cDNA encoding Care were cloned from L.bostrychophila by the methods of RT-PCR and RACE,named Lb est1（GenBank Accession No.:EU854151） and Lb est2（GenBank Accession No.:EU854152）.The complete cDNA of Lb est1 consists of 2049 bp with an ORF of 1713 bp,encoding a protein of 570 amino acids residues with the putative signal peptide of 19 residues.Lb est2 consists of 2525 bp encoding a protein of 617 amino acids residues with the putative signal peptide of 17 residues.Based on the online software ScanProsite,two conserved regions of Care were founded:serine active site（FGGDPNKVTIFGESAG for Lb est1; FGGDPNRITLFGESAG for Lb est2） and the conserved cysteine that compose disulfide bridge （EDCLFLNVFTP for Lb est1;EDCLYLNIYSP for Lb est2）.The sequence homology analysis showed that the two Care genes cloned from L.bostrychophila share low identities to other insect species,but the sequences is highly conserved in active site.1.4 mRNA expression levels of two CarE genes from L.bostrychophilaReal Time PCR analysis showed that the expression level of Lb est2 in DDVP_R and PH_3R strains were 1.91 and 1.42 fold- higher than that of susceptible strain,respectively,and they were significantly different by one way ANOVA（P＜0.05）.The expression levels of two Care genes after treated by DDVP or PH₃ were also significantly higher than control（P＜0.05）.The expression level of Lb est1 gradually decreased with the growth of the psocid,and lowest level was detected at adult stage.In contrast,the expression levels of Lb est2 in nymphal stages are lower than that in adult stage.2 Gene cloning of AChE and mRNA expression level in L.entomophila2.1 Gene cloning and sequence analysis of AChE in L.entomophilaTwo full length cDNA encoding AChE were cloned from L.entomophila by the methods of RT-PCR and RACE,named Le ace1（GenBank Accession No.:EU854149） and Le ace2（GenBank Accession No.:EU854150）.The complete cDNA of Le ace1 consists of 1958 bp with an ORF of 1890 nucleotides,encoding a protein of 629 amino acids residues.Le ace2 consists of 2171 bp with an ORF of 1914 bp,encoding a protein of 637 amino acids residues with the putative signal peptide of 20 residues.The identity of two AChEs is low（35.73%）.The predicted amino acids of two genes from L.entomophila both contained all typical residues of AChE family by sequence alignment, such as the catalytic triad and the oxy-anion hole.Homology modeling of 3-D structure of two AChE from L.entomophila were constructed using H.sapiens（1p0i:A） native BuChE structure and Drosophila melanogaster（1d×4:A） native AChE structure as templates,respectively,by SWISS-MODEL.The catalytic triad were found and denoted in the 3-D structure of AChE from L. entomophila referring to T.californica.2.2 Gene cloning ofβ-actin and mRNA expression levels of two AChE genes from L. entomophilaBecause no reference gene has ever been used in Real Time PCR for L.entomophila in GeneBank,a fragment ofβ-actin gene was cloned from L.entomophila（GenBank Accession No.: FJ041117）.It consists of 822 bp encoding a protein of 273 amino acids residues.The deduced amino acid sequence possesses a high homology toβ-actin from other species reported in GenBank.Real Time PCR analysis showed that Le ace1 expressed 1.6 fold higher than Le ace2 in L.entomophila. The expression levels of the two ace genes after treated by aldicarb or malathion were significantly higher than control（P＜0.05）.3 Gene cloning of AChE and phylogenetic analysis in L.decolorThe full length of Ld ace2（GenBank Accession No.:FJ647187） and fragment of Ld ace1 （GenBank Accession No.:FJ647186） were cloned from L.decolor.The complete cDNA of Ld ace2 consists of 2111 bp with an ORF of 1917 bp,encoding a protein of 638 amino acids residues.The mature protein has a calculated molecular weight of 71.9 kDa with an isoelectric point of 4.7.All the typical residues of AChE family were found in the deduced amino acid sequence of Ld ace2 by sequence alignment.Homology modeling of 3-D structure were constructed using D.melanogaster （1d×4:A） native AChE structure as template.The fragment of Ld ace1 contains 1616 bp encoding 500 amino acid residues.The amino acid sequence has a high identity to ACHE1 from L. bostrychophila and L.entomophila.It includes all the typical residues of AChE family except one conserved cysteine that composes disulfide bridge.The phylogenetic tree of 50 AChE amino acid sequences from 31 species were constructed by MEGA4.1.The results showed that all AChE genes were divided into four distinct main groups: Insect TypeⅠAChE Gene,Vertebrate AChE Gene,Insect TypeⅡAChE Gene and Nematode AChE Gene.The 6 AChE Genes from L.bostrychophila,L.entomophila and L.decolor were demarcated into Insect TypeⅠAChE Gene and Insect TypeⅡAChE Gene.4 Gene cloning of nAChR and mRNA expression level in L.bostrychophilaTwo nicotinic acetylcholine receptor（nAChR） subunit genes,Lb al and Lb a8,were cloned from the psocid L.bostrychophila.The full length cDNAs of Lb a1（GenBank Accession No.: EU871527） and Lb a8（GenBank Accession No.:EU871526） consist of 2025 and 1763 bp, respectively,and contain ORFs of 1644 and 1608 bp encoding 547 and 535 amino acids, respectively.The two subunits shared high identities to those from other insect species,though they share only 56%identity in amino acid sequence.Both genes have features typical of members of nAChR family,such as the long N-terminal extracellular domain and the four hydrophobic transmembrane domains（TM1-4）.The large N-terminal domain of the two nAChR subunits also contains the dicysteine loop（cys-loop） consisting of two disulphide-bond,and the ACh-binding-site-forming regions（loops A-F）.They contain both the two adjacent cysteines which was unique toα-type subunit.The dendrogram shows that the proteins deduced by Lbα1 and Lbα8 belong to theα1-type andα8-type subunits,respectively.Lbα1 possesses a total of 15 potential phosphorylation sites and two potential N-glycosylation sites,while Lbα8 shares 9 potential phosphorylation sites and one potential N-glycosylation site.Quantitative real-time PCR analysis showed that Lbα1 expresses 2.03,2.55,4.77,6.54 and 5.28 fold higher than Lbα8 in the first,second,third,fourth stadium and adult,respectively.The highest expression level of both Lbα1 and Lbα8 was detected at adult stage and lowest was at third and fourth stadium,respectively.There was a stable and relatively low expression level for Lbα1 and a progressive expression pattern for Lbα8 among the 1st,2nd,3rd,and 4th nymphal stages.5 Development and reproduction of L.decolor as a function of temperatureThe developmental rate,survival,intrinsic rate of increase（r_m）,net reproductive rate（R₀）,mean generation time（T）,and population doubling time（t） of L.decolor population were evaluated at eight constant temperatures.The female of L.decolor had four stadia in total nymphs,while the male only had three.Between 20 and 37.5℃,the female developmental period from egg to adult varied from 46.2 d at 20℃to 16.1 d at 35℃,and the male varied from 41.8 d at 20℃to 13.6 d at 35℃,but the high temperature（37.5℃） caused a decline in developmental rate of immature stage of L.decolor.The survival rate from egg to adult was highest（57.3%） at 32.5℃,and lowest（19.0%） at 37.5℃.L. decolor reproduced most eggs（130.4） at 32.5℃and the fewest（24.7） at 37.5℃.The populations reared at 32.5℃expressed the largest r_m（0.0609）,R_o（16.61） and shortest t（11.39 d）.The Weibull frequency distribution suggested a good fit to the data set of age-specific survivorship for all test temperatures.The populations reared at 20-32.5℃have a typeⅠsurvivorship curve（c＞1.0）; whereas populations reared at other temperatures reveal a typeⅢsurvivorship curve（c＜1.0） based on Weibull frequency distribution.In summary,the molecular characteristics of esterases（ACHE and CarE） from three poscid species were studied systematically here.The fact that both two ace genes exist in poscids provides more evidence that two AChE genes are widely distributed in insects.The above results not only fasten the acquaintance of psocid molecular toxicology and enrich the content of evolution and genetic variation,but also contribute to development of molecular diagnostic technique for psocid resistance in field and pave the way for designing new insecticides and develop new strategies for pest management.更多还原