【作者】 修伟明；

【导师】 董双林；

【作者基本信息】 南京农业大学 ， 农业昆虫与害虫防治， 2007， 博士

【摘要】 昆虫在长期进化过程中形成了高度灵敏的嗅觉系统，并以此来感受自然环境中的各种化学信息，产生相应的生理和行为反应，如寻找配偶、食物源和栖息场所、产卵行为、逃避危险或者不适合的栖息地和寄主等，因此嗅觉系统对于昆虫的生命活动至关重要。将昆虫嗅觉应用于害虫防治，已成为害虫管理的一个重要手段，并将在未来害虫治理中起到越来越重要的作用。甜菜夜蛾和斜纹夜蛾是世界性分布的重要农业害虫，危害很多种粮食和蔬菜作物，生产上主要依靠化学防治，但由于害虫抗药性以及蔬菜等作物的无公害防治要求，迫切需要寻求新的防治技术。这两种夜蛾雌虫性信息素组分已得到鉴定，并成功用于田间种群的预测预报，但将性信息素直接用于害虫的大量诱杀或交配干扰，防治效果并不理想。因此，深入研究蛾类昆虫性信息素通讯的分子机制，弄清性信息素感受相关蛋白及其功能，必将有助于针对特定嗅觉蛋白设计和开发更为高效的两性通讯的调控技术，服务于蔬菜和粮食的无公害生产。本文对甜菜夜蛾和斜纹夜蛾性信息素感受相关蛋白进行了研究，主要结果如下：1甜菜夜蛾信息素结合蛋白基因的克隆和序列分析通过比较几种已发表夜蛾科昆虫的信息素结合蛋白(PBPs)氨基酸序列，设计合成一对简并性引物，利用反转录多聚酶链式反应(RT-PCR)技术从甜菜夜蛾(Spodoptera exigua Hübner)雄虫触角扩增得到2个分别为275bp和281bp的cDNA片段SexigPBP1和SexigPBP2。随后，利用cDNA末端快速扩增(RACE)技术进行两个cDNA全序列克隆，SexigPBP1 (Genbank登录号：AY540316)cDNA全序列为1，077bp，SexigPBP2 (Genbaak登录号：AY545636)为819bp。S．exigua的2个PBP基因推导的氨基酸序列与已知其它鳞翅目昆虫PBP具有较高的同源性，但彼此间的同源性为44％。以基因组DNA为模板进行扩增后，发现S．exigua的2个PBP基因具有相同的intron／extron结构，各含有2个内含子，并且内含子具有典型的GT-AG结构。SexigPBP1在Glu45和Leu46之间插入一个87 bp的内含子：在Asp105内插入第二个内含子，长142bp。SexigPBP2在Glu49和Met50之间插入一个295 bp的内含子；在Asp110内插入第二个内含子，长644bp。RT-PCR结果表明SexigPBP1和SexigPBP2在雌雄虫触角内均有表达，但在其他组织中不表达。进一步用实时定量PCR技术比较了雌雄蛾触角内两个PBP基因的表达水平，结果发现，雌蛾触角内的SexiegPBP1和SexigPBP2的表达水平仅为雄蛾的39％和73％。2斜纹夜蛾信息素结合蛋白基因的克隆和序列分析利用RT-PCR和RACE技术，从斜纹夜蛾(S．litura(Fabrieius)，)雄蛾触角扩增得到2个信息素结合蛋白(PBPs)cDNA全序列：SlitPBP1和SlitPBP2。SlitPBP1(Genbank登录号：DQ004497)全序列为1，085 bp，编码164个氨基酸，N-端头23个氨基酸为预测的信号肽，其后的成熟蛋白由141个氨基酸组成，预测分子量为16，285 Da，等电点为5.14。SlitPBP2 (Genbank登录号：DQ114219)全长804 bp，编码170个氨基酸，最初27个氨基酸为信号肽，143个氨基酸组成的成熟蛋白预测分子量为15，998 Da，等电点为4.92。2个PBP基因推导的氨基酸序列与已知其它鳞翅目昆虫PBP具有较高的同源性，两者彼此之间的同源性为45％。SlitPBP1和SlitPBP2内各含有2个内含子，并且内含子具有典型的GT-AG结构。SlitPBP1在Glu45和Leu46之间插入一个76 bp的内含子；在Asp105内插入第二个内含子，长104 bp。SlitPBP2在Glu49和Met50之间插入一个437 bp的内含子；在Asp110内插入第二个内含子，长1，251 bp。可见，PBP2的内含子比PBP1长得多，同一个基因第二个内含子明显比第一个长。RT-PCR结果表明SIitPBP1和Slit PBP2仅在雌雄虫触角内表达。进一步用实时定量PCR技术比较了雌雄虫触角内SlitPBP1和Slit PBP2的表达水平，结果发现，雌蛾触角内的SlitPBP1和SlitPBP2的表达水平仅为雄蛾的2.1％和7.0％。搜集现已报道的鳞翅目昆虫PBP，利用Mega3.1软件进行系统进化分析，结果显示夜蛾科昆虫的PBP被清楚地分为三个组，本研究发现的SexigPBP1和SlitPBP1同属于第二组(Group2)，SexigPBP2和SlitPBP2同属于第三组(Group3)。3甜菜夜蛾信息素结合蛋白基因的原核表达及蛋白纯化在成功克隆甜菜夜蛾2个PBP基因的基础上，将SexigPBP1和SexigPBP2构建到原核表达载体pET-30a(+)中，在大肠杆菌BL21(DE3)中成功表达，表达产物经SDS-PAGE检测，所表达的两个重组蛋白约22kD，主要以包涵体形式存在。超声波破碎离心后，用8M尿素对包涵体进行溶解，经镍离子亲和层析柱一步法纯化得到的PBP的纯度非常高，并且回收率也很高。为了避免N-端多余序列的干扰作用，利用肠激酶对已纯化的目的蛋白进行专一性酶解，酶解产物再经亲和层析纯化，得到已除去N-端标签的目的蛋白(约16kD)，为进一步研究PBP与性信息素组分之间的作用打下基础。利用常规免疫方法，用重折叠的目的蛋白SexigPBPl免疫Babl／c小鼠获得较高效价抗体，为进行下一步实验提供材料。4甜菜夜蛾和斜纹夜蛾嗅觉受体基因的克隆和序列分析高度变异的昆虫嗅觉受体家族中有一类在不同目昆虫中非常保守的嗅觉受体，本文通过比对几种已发表昆虫的嗅觉受体(ORs)氨基酸序列，设计合成一对简并性引物，分别在甜菜夜蛾和斜纹夜蛾触角内克隆到292 bp和1，063bp的cDNA片段，命名为SexiOR2和SlitOR2。利用cDNA末端快速扩增(RACE)技术进行两个cDNA全序列克隆，SexiOR2(Genbank登录号：AY862142)全长为1，906 bp，阅读框架全长1，419 bp，编码473个氨基酸，预测分子量为53，303 Da，等电点为8.60。SlitOR2(Genbank登录号：DQ845292)全长为2，483 bp，阅读框架全长1，419 bp，编码473个氨基酸，预测分子量为53，267 Da，等电点为8.63。SlitOR2的3′非编码区(937 bp)要比SexiOR2的(350 bp)长很多。经预测氨基酸序列中具有典型的G蛋白偶联受体(GPCR)的七个跨膜区域。SexiOR2和SlitOR2与已知其它昆虫嗅觉受体的同源性都在50％以上，并且两者之间的同源性高达98％。系统发育分析表明，已知的鳞翅目昆虫嗅觉受体蛋白独立形成一组。本论文利用分子生物学技术，成功克隆出与甜菜夜蛾和斜纹夜蛾性信息素感受有关的两类蛋白，并进行了系统进化等相关分析。在此基础上，用原核表达系统表达并纯化了甜菜夜蛾的2个PBP，并制备了甜菜夜蛾PBP1的抗体。研究结果为弄清这两种夜蛾性信息素的感受机制打下了基础，并为研制和开发新型、高效的两性通讯阻断剂提供了依据。更多还原

【Abstract】 In insects, the olfactory systems have been evolved highly sensitive, enabling detect and discriminate among a diverse array of volatile chemicals that stimulus insect behavioral responses such as mate-seeking, location of food and habitat, oviposition. Olfactory systems play crucial roles in insect survival and reproductive success. The olfactory-based control strategies have developed an important means for pest control. The beet armyworm, Spodoptera exigua Hübner and common cutworm S. litura (Fabricius) (Lepidoptera: Noetuidae), are severe pests of various agricultural crops. Their sex pheromone of female moths have been identified as complex blends, and successfully used in pest population monitoring. However, it is not satisfactory to its use in pest control except with means of mass-traping and mating-disruption. Therefore, it is very important to gain insight on the molecular mechanism of the perception of female sex pheromone by the male. With the insights on the molecular machemism, it will be facilitated for molecular designing of specific regulator to these key olfactory proteins, and for further development of more effective pest behaviorally interfereing techniques. Here, we report the molecular identification and sequence analysis of PBP and odorant receptor genes from S. exigua and S. litura, and further experession and purification of the PBP protein. The main results are as follows.1. Molecular characterization and sequence analysis of PBP from S. exiguaPCR performed with a pair of degenerate primers designed on the conserved amino acid regions of FWK(R)EG(E)Y and HE(D)LN(K)WA in PBPs of other insects, afforded two cDNA products of 275 and 281 bp, respectively. They were called SexigPBP1 and SexigPBP2, respectively. A Rapid Amplification of cDNA Ends (RACE) procedure was employed to obtain full-length sequences of PBP1 and PBP2 from S. exigua. The two PBP sequences of SexigPBP1 and SexigPBP2 were deposited in GenBank with the accession numbers of AY540316 and AY545636, respectively. The full-length sequences of SexigPBP1 and SexigPBP2 are 1,077 and 819 bp, respectively. SexigPBP1 cDNA contains a 492 bp open reading frame that encodes a 164 amino acid protein. SexigPBP2 cDNA contains a 510 bp open reading frame that encodes a 170 amino acid protein. The two PBPs from S. exigua share low similarity (44% identity), but are very similar to PBPs of the same group from other insect species, respectively. In order to ascertain the intron/exon structure of the genes, the two PBP genes were further cloned and sequenced from the genomie DNA preparation. Both SexigPBP1 and SexigPBP2 genes contain two introns, and present a similar intron/exon structure. The ends of the introns have a typical GT-AG structure. The two introns in SexigPBP1 are located between Glu45 and Leu46 (intronl, 87 bp) and inside the codon for Aspl05 (intron2, 142 bp). The introns in SexigPBP2 are located between Glu49 and Met50 (intronl, 295bp) and inside the eodon for Asp110 (intron2, 644 bp). To investigate the presence of PBPs in different tissues of S. exigua, reverse transeription-polymerase chain reaction (RT-PCR) experiments were performed using specific primers. SexigPBP1 and SexigPBP2 are only expressed in S. exigua antennae. Real-time PCR was performed to compare the transcript levels of SexigPBP1 and SexigPBP2 from antennae of male to female moths. The transcription levels of PBP genes from antennae were higher in male moths than in females. The amounts of SexigPBP1 and SexigPBP2 mRNA in female antennae were about 39% and 73%, respectively, relative to those in male antennae.2. Cloning and sequence analysis of PBP from S. lituraSimilarly as in S. exigua, with RT-PCR technique, two cDNA fragments (275 and 281 bp) were cloned. They were named SlitPBP1 and SlitPBP2, respectively. Their full lengths were obtained using RACE procedure. The isolated cDNA clone encoding SlitPBP1 (GenBank accession numbers DQ004497) was 1,085 bp long and contained a 492 bp open reading frame for a polypeptide of 164 amino acids. The initial 23 amino acids are predicted as a signal peptide followed by the native protein of 141 amino acids with a molecular mass of 16,285 Da, and an isoelectric point of 5.14. The isolated cDNA clone encoding SlitPBP2 (804 bp) (GenBank accession numbers DQl14219) comprised the complete PBP precursor consisting of a 27 amino acids long signal peptide followed by the native protein of 143 amino acids with a molecular mass of 15,998 Da and a isoelectric point of 4.92. Both SlitPBP1 and SlitPBP2 genes also contain two introns from the genomic DNA preparation. The two introns in SlitPBP1 are located between Glu45 and Leu46 (intronl, 76 bp) and inside the codon for Asp105 (intron2, 104 bp). The introns in SlitPBP2 are located between Glu49 and Met50 (intronl, 437 bp) and inside the codon for Asp110 (intron2, 1251 bp). The ends of the introns have a typical GT-AG structure. The two PBPs from S. litura share low similarity (45% identity), but are very similar to PBPs of the same group from other insect species, respectively. RT-PCR experiments show SlitPBP1 and SlitPBP2 are only expressed in S. litura antennae. The transcription levels of PBP genes from male and antennae were compared with Real-time PCR. The amounts of SlitPBP1 and SlitPBP2 mRNA in female antennae were about 2.1% and 7%, respectively, relative to those in male antennae. Phylogenetic analysis showed that noctuid PBPs were classified into three distinct groups (Group 1-3), and SexigPBP1, SlitPBP 1 and SexigPBP2, SlitPBP2 belong to two different groups (Group 2 and Group 3), respectively.3. Bacterial expression and purification of PBPs from S. exiguaBased on cloned SexigPBP information, SexigPBP1 and SexigPBP2 were constructed into bacterial expression vector pET-30a(+) fused to N-terminal His-tag sequence in frame and successfully overexpressed in E. coli BL21 (DE3), mostly present as inclusion bodies. The two recombinant proteins showed an obvious band at about 22kD through sodium dodecyl sulfate-polyacrylamide gel eleetrophoresis (SDS-PAGE) analysis. The induced cells were disrupted by sonication on ice. The isolate inclusion bodies were solubilized in 8 Murea. Extracts were purified with a nickel affinity column. Cleavage of the fusion protein, with enterokinase and further purification led to an expected molecular weight protein (about 16kD). In adition, the purified SexigPBP1 was refolded and used to immunize mouse, and the antibodies with higher immuo-effeiceney were obtained and tested by enzyme-linked immunosorbent assay.4. Cloning and sequence analysis of odorant receptor genes from S. exigua and S. iituraBy RT-PCR method with a pair of degenerate primers designed against an alignment of several known insect odorant receptor sequences. Two cDNA fragments of odorant receptor gene, SexiOR2 and SlitOR2, were cloned and sequenced from the antennae of male S. exigua and S. litura. Their full lengths were obtained using RACE procedure. The full-length sequence of SexiOR2 (Genbank accession number AY862142) was 1,906 bp, contains a 1,419 bp bp open reading frame that encodes a 473 amino acid protein with a molecular mass of 53,303 Da and an isoelectrie point of 8.60. The full-length sequence of SlitOR2 (GenBank accession numbers DQ845292) was 2,483 bp long and also contained a 1,419 bp open reading frame for a polypeptide of 473 amino acids with a molecular mass of 53,267 Da and an isoelectric point of 8.63. SexiOR2 and SlitOR2 included seven putative transmembrane domains, which is the typical characteristic of G protein-coupled receptors. The sequence analysis indicated that the deduced amino acid sequences of SexiOR2 and SlitOR2 shared high identity with olfactory receptors from other previously reported insects (50%) and the homology between SexiOR2 and SlitOR2 reached 98%. Phylogenetic analysis showed that Drosophila melanogaster Or83b and its Orthologs reported from other insects were classified into three distinct groups, and noctuid olfactory receptors formed a group.In this paper, we describe the identification and characterization of pheromone reception related proteins from the antennae of S. exigua and S. litura, and report about the comparison and evolution of these proteins of orthologous proteins. Based on cloned SexigPBP information, bacterial expression vectors of SexigPBP1 and SexigPBP2 were constructed and successfully expressed. The recombinant proteins were purified by affinity chromatography. Furthermore, the antiserum was raised with the purified SexigPBP1. These results could be used to explore the molecular mechanism of pheromone reception in S. exigua and S. litura, and helpful for providing theoretical evidences in designing and developing new effective attractant or deterrent.更多还原