【作者】 苏宏华；

【导师】 郭予元；

【作者基本信息】 中国农业科学院 ， 农业昆虫与害虫防治， 2006， 博士

【摘要】 棉铃虫是许多国家农业上的重要害虫。研究表明,昆虫的嗅觉在处理来自环境的化学信号过程中起着非常重要的作用,从而使昆虫能够觅食,寻找配偶、产卵场所、寄主和猎物,及发现捕食者等。昆虫的气味识别过程非常复杂,有多种蛋白参与。迄今为止,昆虫的嗅觉识别机制研究取得了一些可喜的进展,但是对整个嗅觉信号传导途径了解得非常少。清楚阐明昆虫的嗅觉信号传导会为研制高效的引诱剂或杀虫剂提供理论依据,为有效防治棉铃虫提供新的思路与途径,延缓或降低棉铃虫对Bt棉花抗性演化的速度。本文利用RACE技术结合PCR克隆了Gq蛋白α亚基基因;采用半定量RT-PCR研究了Gq α和SNMP的两个基因在棉铃虫体内的时空表达情况;对两个基因进行原核表达和纯化;利用酵母双杂交技术研究已经克隆的Gq α和SNMP是否互作,并筛选与Gq α相互作用的蛋白。本研究所取得的结果如下: (1)利用PCR结合RACE技术从棉铃虫触角中首次克隆了G蛋白α亚基基因,经过BLAST搜索同源序列发现其属于G蛋白α亚基的q家族。已被GenBank数据库收录,登录号AY957405,命名为Gq a Harm。Gq α Harm与已报道的昆虫和其他无脊椎动物的Gq蛋白α亚基相似性在90%以上。与近缘种甘蓝夜蛾Mamestra brassicae Gq α的相似性高达96.88%;与家蚕Bomyx mori Gq α的相似性高达97.73%;与果蝇(Drosophila melanogaster)Gq α的相似性高达89.52%;与线虫Caenorhabditis elegansGq α的相似性高达81.69%;与加勒比龙虾Panulirus argus Gq α的相似性高达87.25%。 (2)半定量RT-PCR研究表明,Gq α Harm在棉铃虫雌雄成虫触角中表达,在头、胸、腹、足和翅中也有表达,并且表达量差异不显著;此外,在喙、下颚须和下唇须中也有表达;在卵、幼虫、蛹和成虫体内也都有表达;在卵中的表达量最高,而在成虫中的表达量最低,在幼虫和蛹的前、中、后期的表达量差异不大。研究表明Gq α Harm是一种普遍存在的蛋白。棉铃虫Gq α Harm的这种组织分布广泛性,可能预示其参与细胞的多种信号传导途径。 (3)棉铃虫感觉神经元膜蛋白基因序列法国人已经报道(Jacquin-Joly,E.andFrancois,M.-C.,2003 GenBanK,AF462067)。利用特异性引物从本实验室棉铃虫触角中克隆了一条全长1690bp的cDNA序列,感觉神经元膜蛋白(SNMP)阅读框全长1572bp,编码523个氨基酸残基,序列中有2个跨膜区,具有昆虫感觉神经元膜蛋白的典型特征。SNMP与已报道的其它昆虫的感觉神经元蛋白的氨基酸序列有很高的相似性。半定量RT-PCR研究结果显示,SNMP在棉铃虫中不仅在触角中表达,也在去掉触角的头、足中表达。但是在触角中的表达量最高,在雌雄触角中的表达量差异不显著。在棉铃虫的味觉器官如:喙,下颚须和下唇须中也有表达。在卵、蛹和成虫体内也都有表达,在卵中表达量相对较低。SNMP在棉铃虫体内的时空表达情况表明:SNMP不仅参与棉铃虫的嗅觉识别,而且还参与棉铃虫的味觉识别。更多还原

【Abstract】 Cotton bollworm (Helicoverpa armigera) is a dominant pest for agriculture in many countries. For insects, olfaction plays an essential role in processing chemical signals from the environment, leading to the detection of food, reproductive partners, oviposition sites, host, prey or predators. Odor detection is a complex procedure, in which many proteins are involved. Till now, some exciting progresses have been made in the olfaction recognition mechanism for insects, but the complete olfactory siganl transduction pathway is pooly understood. Elucidation of the olfaction signalling will provide the theoretical foundation for the development of the effective attractant and deterrent, and the fresh ideas and approaches for potent control of cotton bollworm, which can slower the revolution of its resistance to the Bt. cotton. In this paper, a Gq a subunit gene was cloned by RACE techniques and PCR. Temporal and spacial expression patterns of Gqa and SNMP in cotton bollworm were studied by semi-quantitive RT-PCR. Recombinant Gqa and SNMP were obtained by prokaryotic expression and purified by affinity chromatography. The interaction between the two cloned genes was determined by Yeast Two-Hybrid System and screening for related proteins to Gqo from H.armigera antennae-cDNA library were conducted with the same technique. The main results are as follows:(1) G protein a subunit was cloned for the first time from the antennae of Helicoverpa armigera by RT-PCR and (3’/5’)-RACE techniques. It belonged to the Gaq family. The cDNA sequence was deposited in GenBank and the accession number is AY957405 and named as GqaHarm. The deduced amino acid sequence of GqaHarm shared high identity (>90%) with reported Gqa from other insects and invertebrates. The homology with Bombyx mori, its close sib population Mamestra brassicae, Drosophila melanogaster, Caenorhabditis elegans and Panulirus argus was up to 97.73%, 96.88%, 89.52%, 81.69%, 87.25% respectively.(2) Tissue-and stage-specific transcriptive levels were determined by semi-quantitative RT-PCR analysis and the results showed that Gqa was transcripted in multiple tissues and at different stages. Gqa was not only expressed in antenna, but also in the head without antenna, thorax, leg and wing of H. armigera. There was no obvious difference among the expression in the tissues. Moreover, it was also expressed in labial palpus, maxillary palpus and proboscis. Gqa was expressed in egg with the highest level, and in imago with the lowest level;and there was no significant diffenrence among the expression in different stages for the larva/and the pupae. The spacial and temporal expression pattern indicated that GqaHarm was a/ ubiquitous protein, which suggested that it might be involved in diverse signalingpathways.(3) Sensory neuron membrane protein (SNMP) of Helicoverpa armigera was reported by French Jacquin-Joly,E. and Francois,M.-C. in the year 2003 (GenBanK accession number: AF462067). A full-length cDNA encoding SNMP, was cloned by using the specific primers to the cDNA template from antennae of Helicoverpa armigera reared in our lab. The open reading-frame of SNMP was 1572bp, encoding 523 amino acid residues. There were two putative transmembrane domains, which is the typical characteristic of SNMPs. And it also shared high identity with sensory neuron membrane proteins from other insects. Semi-quantitative RT-PCR analysis showed that SNMP was not only expressed in antenna, but also in the head without antenna and leg of//, armigera. However, its expression in antenna was much higher than that in the head without antenna and leg. There was no obvious difference between the expression in the male and female antenna. Moreover, it was also expressed in labial palpus, maxillary palpus and proboscis which are the taste organs. SNMP was expressed in egg, the 10tb-day pupae and imago and expressed relatively low in egg. The spacial and temporal expression pattern indicated that SNMP may play roles in not only the odor detection but also the taste detection.(4) GqaHarm and SNMP genes were subcloned into prokaryotic expression vector pET-21b respectively. Bacteric expression vectors of target genes were successfully constructed and the target proteins were successfully expressed in E.coli through the inducement of IPTG. However , the expression product was insoluble inclusion bodies, which were solubilized and refolded . Then the recombinant proteins were purified by affinity chromatography. Pure Gqa protein was obtained , but SNMP was not purified successfully by the same purification method.(5) Interaction proteins were screened from the cDNA library of Helicoverpa.armigera antennae with cloned a subunit as bait protein by Yeast Two-Hybrid System. Forty-three positive clones were identified by SD/-Ade/-His/-Leu/-Trp/X-a-GaI and sequenced. Through GenBank blast, the cDNA inserts were highly similar to such mRNAs as Helicoverpa armigera pheromone binding protein (PBPHarm), Heliothis virescens pheromone binding protein-2 (PBPHvir2), Plutella xylostella glyceraldehyde-3-phosphate dehydrogenase, Spodoptera frugiperda ribosoma! protein S20, Helicoverpa armigera cytochrome oxidase subunit I gene, tRNA-Leu gene and cytochrome c oxidase subunit II gene,, Spodopterafrugiperda allatotropin neuropeptide precursor(AT2 gene), Bombyx^mori translationally controlled tumor protein (TCTP) and humoral lectin prepropeptfa/. Identification of these valuable clones provided substaintial foundation for furMef*. study of the function of G protein a subunit in signaling pathway of Helicoverpaarmigera.(5) The interaction between cloned Gq a subunit and SNMP was determined by Two-Hybrid System and no positive clones were obtained. One of the possible reasons is that proteins with weak affinities may escape detection or there is no interaction between the two proteins in Helicoverpa armigera. SNMP is presumed to interact with some a subunit of other G protein a subunit famlies, which needs the further studyIn this paper, G-protein a subunit was cloned for the first time from the antennae of Helicoverpa armigera. The accession number in GenBank is AY957405. The study on tissue- and temporal expression pattern of Gqa and SNMP indicated that G protein a subunit was a ubiquitous protein in Helicoverpa armigera and might be involved in diverse signaling pathways. The temporal and spacial expression pattern of SNMP suggested that SNMP was not antennae-specific, which was involved in both olfactory detection an taste function. These results of two-hybrid screening indicated that G protein a subunit could directly interact with pheromone binding protein, which had some roles in odor signal transduction. G protein a subunit could not interact with SNMP in the two hybrid system.There is no internal and oversea publication concerned with above studies on G proteins in Helicoverpa armigera. These achievements provide substaintial foundation for further research of G proteins and G-protein signaling pathways in cotton bollworm. It also has important theoretic and practical significance on going deep into interaction between insect and host plant, pest-resistant mechanism of plant and exploring new control method.更多还原