【作者】 邵刚锋；

【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， 2009， 硕士

【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触角内两类气味降解相关酶类进行了研究,主要结果如下：1甜菜夜蛾P450基因的克隆、序列分析及组织表达谱分析通过比较几种已发表夜蛾科昆虫的细胞色素P450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 Hubner)雄虫触角扩增得到2个分别为444bp和441bp的cDNA片段,由P450命名委员会命名为CYP4L15和CYP4L16。S. exigua的2个P450基因推导的氨基酸序列与已知其它鳞翅目昆虫P450基因具有较高的同源性,其中CYP4L15和甘蓝夜蛾CYP4L4基因相似性高达94%；CYP4L16和甘蓝夜蛾CYP4L4基因相似性高达92%。2个基因间的相似性为96%。RT-PCR结果表明,CYP4L15和CYP4L16在雌雄虫触角内均有表达,且表达量相当,说明该P450基因没有性特异性。CYP4L15基因在雄蛾各个组织中都有表达；CYP4L16除在雄蛾腹部没有明显表达外,在其它组织中均表达；两个基因在翅内的表达量均较低。根据上述结果,推测2个基因可能参与了昆虫的化学感受；而CYP4L15可能还参与对进入消化道的植物次生物质的代谢等。2斜纹夜蛾P450基因的克隆、序列分析及组织表达谱分析通过比较几种已发表夜蛾科昆虫的细胞色素P450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)技术从斜纹夜蛾(S. litura (Fabricius))雄蛾触角扩增得到3个细胞色素P450 cDNA片段,由P450命名委员会命名为CYP4L19、CYP4L20和CYP4L21。3个基因推导的氨基酸序列与已知其它昆虫P450具有较高的同源性,CYP4L19和甘蓝夜蛾CYP4L4基因相似性达到94%；CYP4L20和甘蓝夜蛾CYP4L4基因相似性达到92%；CYP4L21和甘蓝夜蛾CYP4L4基因相似性达到92%。CYP4L19和甜菜夜蛾细胞色素P450基因CYP4L15和CYP4L16的相似性分别为97%和95%；CYP4L20和甜菜夜蛾细胞色素P450基因CYP4L15和CYP4L16的相似性分别为95%和93%；CYP4L21和甜菜夜蛾细胞色素P450基因CYP4L15和CYP4L16的相似性分别为96%和93%；3个斜纹夜蛾P450 cDNA片段间的氨基酸序列的相似性互为97%。RT-PCR结果表明3个基因在雌雄蛾触角内都有表达,且表达量相当,说明3个基因在雌雄蛾触角内担负相似的功能,并具有相似的重要性。除CYP4L21在雄蛾翅内没有表达外,3个基因在所有测定的组织中均表达,以触角、腹部和足中的表达量较高,而头、胸及翅中的表达量较低。因此,同甜菜夜蛾的2个P450基因类似,3个斜纹夜蛾P450基因可能担负对外源气味物质、食物中的有毒物质及杀虫剂的代谢作用。3甜菜夜蛾和斜纹夜蛾触角酯酶基因克隆、序列及组织表达谱分析设计合成一对简并性引物,在甜菜夜蛾和斜纹夜蛾触角内分别克隆到1个长为393bp的cDNA片段,命名为SexiAE和SlitAE。SexiAE与已知其它昆虫具气味降解活性酯酶的同源性较高,其中和甘蓝夜蛾气味降解酯酶相似性最高为54%。SlitAE与已知甘蓝夜蛾气味降解酯酶和海灰翅夜蛾触角酯酶的相似性最高,均为51%,SexiAE和SlitAE相似性为67%。RT-PCR结果表明,SexiAE和SlitAE在甜菜夜蛾和斜纹夜蛾雌雄触角内均有表达,且表达量相当,不具有性特异性,推测2个酯酶基因可能参与了包括性信息素组分在内的多种气味物质的降解。SexiAE在甜菜夜蛾雄虫各个组织内都有表达,但在去触角的头部较低。SlitAE在斜纹夜蛾雄虫触角、腹部表达量较高,在足内表达微弱,在其它组织内没有表达。2个基因在腹部等非化感组织的表达,说明它们可能还担负降解气味物质以外的功能。由于SexiAE较SlitAE具有更宽的组织表达谱,说明前者可能参与更多的生理功能,其底物谱也可能更宽。本文利用分子生物学技术,成功克隆出甜菜夜蛾和斜纹夜蛾几个P450基因和酯酶基因,并进行了序列及组织表达谱等相关分析。研究结果为进一步明确两种夜蛾的气味感受机制,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 evolved to be highly sensitive, enabling detection and discrimination among a diverse array of volatile chemicals, and thus leading to various behavioral responses such as mate-seeking, location of food and habitat, oviposition. Therefore, olfactory systems play crucial roles in insect survival and reproductive success. The olfactory-based control strategies have developed as an important means for pest control. The beet armyworm, Spodoptera exigua (Hiibner) and common cutworm, S. litura (Fabricius) (Lepidoptera: noctuidae), 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 use it in pest control neither by means of mass-trapping nor mating-disruption. Therefore, it is very important to gain insight on the molecular mechanism of the perception of female sex pheromone by the male. The insights into the molecular mechanism will facilitate molecular designing of specific regulator to olfactory related proteins, and for further development of more effective pest behaviorally interfering techniques. Here, we report the molecular identification and sequence analysis of P450 and antennal esterase genes from S. exigua and S. litura, and further tissure expression patterns of these genes. The main results are as follows.1. Molecular characterization and Tissue expression analysis of P450 genes from S. exiguaPCR was performed with a pair of degenerate primers designed on the conserved amino acid regions in P450 of other insects, afforded two cDNA products of 444 and 441 bp, respectively. They were named CYP4L15 and CYP4L16, respectively. Alignment with other p450 genes putatively involved in odorant degradation, high identities were found among these proteins. The identity between gene CYP4L15 and CYP4L4 from M. brassicae was 94%; the identity between CYP4L16 and CYP4L4 was 92%. Two novel genes had a high identity of 96%.To investigate the expression of P450 in different tissues of S. exigua, reverse transcription-polymerase chain reaction (RT-PCR) experiments were performed using specific primers. CYP4L15 and CYP4L16 were expressed in male and female antennae of S. exigua, and got equal expression levels. CYP4L15 expressed in all tissues tested (antennae, head without antennae, thorax, abdomen, feet, wing) in male moth. CYP4L16 expressed in all tissues tested except for the abdomen in male moth. Both CYP4L15 and CYP4L16 were lowerly expressed in wings. Based on these results,2 genes were putatively proposed to involve in chemoreception, with CYP4L15 also in the metabolism of toxical chemicals in the alimentary canal.2. Molecular characterization and Tissue expression analysis of P450 genes from S. lituraSimilarly as in S. exigua, with RT-PCR technique, three cDNA fragments (444bp) were cloned. They were named CYP4L19, CYP4L20 and CYP4L21, respectively. Alignment with the other p450s putatively involved in odorant degradation, high identities were found among these proteins. The identity with CYP4L19 and CYP4L4 was 94%; both CYP4L20 and CYP4L21 have identity with CYP4L4 of 92%. CYP4L19 has identity of 97% and 95% with two P450 genes cloned from S. exigua CYP4L15 and CYP4L16; CYP4L20 has identity of 95% and 93% with CYP4L15 and CYP4L16; CYP4L21 has identity of 96% and 93% with CYP4L15 and CYP4L16. Three novel genes have high identity of 97%.To investigate expression of P450 in different tissues of S. litura, RT-PCR experiments were performed using specific primers. RT-PCR experiments show CYP4L19, CYP4L20 and CYP4L21 were expressed with equal level in male and female S. litura antennae. Six tissues were selected for expression comparison in the male moth, including antennae, head with antennae deduced, thorax, abdomen, feet and wing. CYP4L19 and CYP4L20 were expressed in all tested tissues, with higher amount in antennae, abdomen and feet. CYP4L21 expressed in all tested tissues except for in wing. Three cloned novel genes were proposed to play roles in metabolism of odorant moleculars, toxical chemicals and pesticide entered into alimentary canal with food. 3. Molecular cloning, Characterization and Tissue expression analysis of esterase genes from S. exigua and S. lituraBy RT-PCR method with a pair of degenerate primers designed against an alignment of several known insect odorant degrading esterase animo acid sequences. Two cDNA fragments of antennal esterase gene, SexiAE and SlitAE were cloned and sequenced from the antennae of male S. exigua and S. litura, respectively. Alignment with the other esterase genes that putatively involved in odorant degradation, identities were analysised. SexiAE shared the highet identity of 54% with odorant degrading esterase gene of MbraODE, and SlitAE shared the highest identity of 51% with esterase of MbraODE and SlitEST. Two novel genes shared an identity of 67%.To investigate the expression of SexiAE and SlitAE in different tissues of S.exigua and S. litura, RT-PCR experiments were performed using specific primers. SexiAE and SlitAE were both expressed in male and female antennae of S. exigua and S. litura, respectively. Six tissues were selected for the expression study in the male moth, and these tissues were male antennae, head with antennae deduced, thorax, abdomen, feet and wing. SexiAE was expressed in all tested tissues, but the expression leve in wing was very low. SlitAE was expressed with higher level in antennae and abdomen than that in feet. There was no expression in other tissues tested. The expression pattern of these two esterase genes indicated that 2 esterases may play multiple roles besides odorant metabolism in chemoreception, and that. SexiAE comparing to SlitAE, may has wider substrate spectrum and thus undertake more physiological functions.In conclusion, we described the identification and characterization of odorant reception related enzymes from the male antennae of S. exigua and S. litura, and reported their tissue expression pattern in S. exigua or S. litura. The results could be helpful for both understanding of the molecular mechanism of odorant reception in S. exigua and S. litura, and providing theoretical bases in designing and developing new tactics in pest control.更多还原