斜纹夜蛾与棉铃虫化感基因鉴定及受体功能分析

发布时间：2018-08-10 18:40

【摘要】：作为地球上种类和数量最大的生物类群,昆虫在漫长的进化过程中,形成了高度灵敏的嗅觉感受系统,以此来感受环境中纷繁复杂的气味物质,从而完成寻找配偶、食物、产卵场所和逃避天敌等重要的行为活动,因此研究昆虫嗅觉感受机制具有重要的科学意义。从应用的角度讲,我国长期以来采用化学农药防治害虫,不仅使害虫产生了抗性,防治效果降低,同时农药残留给环境及人类生活带来的安全隐患日益严重并引起格外关注,亟需开发更环保、更安全的害虫防治技术。基于昆虫两性之间高度特异的信息素通讯而设计开发的性引诱剂已经成功应用于一些害虫的防治,但总体效果仍无法达到人们预期,主要原因是对昆虫性信息素或其他气味物质的嗅觉机制还不够了解。昆虫对于气味物质的感受和识别涉及多个蛋白,如气味结合蛋白(Odorant binding protein,OBP)、化学感受蛋白(Chemosensory protein,CSP)、气味受体(Odorant receptor,OR)、离子型受体(Ionotropic receptor,IR)、感觉神经元膜蛋白(Sensory neuron membrane protein,SNMP)和气味降解酶(Odorant degrading enzyme,ODE)等。深入研究这些基因的功能及其在气味识别过程中的作用机制,无疑将有助于开发更高效的害虫防治技术。本研究以重要农业害虫斜纹夜蛾(Spodoptera litura)和棉铃虫(Helicoverpa armigera)为研究对象,结合分子生物学、生物信息学和电生理学技术,重点对OR和SNMP两类基因进行了鉴定及功能分析,主要结果如下:1.斜纹夜蛾性信息素受体基因的克隆、表达及功能研究在鳞翅目昆虫中,根据功能差异一般可以将OR分为性信息素受体(pheromone receptor,PR)和非性信息素受体(non-pheromone receptor,non-PR OR)。根据海灰翅夜蛾(Spodoptera littoralis)报道的4个PR基因,设计特异性引物,从斜纹夜蛾雄蛾触角中克隆并鉴定了4个PR基因片段,进一步通过RACE技术获得其全长cDNA序列,分别命名为SlituOR6(Genbank登录号:KC188666)、SlituOR11(KC188667)、SlituOR13(KC188668)和SlituOR16(KC188669)。组织表达谱显示,4个PR主要在触角中表达,SlituOR6和SlituOR13在雄虫触角特异表达,SlituOR11和SlituOR16在雄虫触角高表达。进一步采用非洲爪蟾卵母细胞(Xenopus oocytes)系统表达并分析4个PRs的功能,其中SlituOR6对次要性信息素组分Z9,E12-14:OAc特异反应(EC_(50)=1.99×10~(-6) M);SlituOR13对次要性信息素组分Z9,E12-14:OAc(EC_(50)=6.109×10~(-6)M)和Z9-14:OAc(EC_(50)=1.184×10~(-6)M)反应比较强烈,对主要性信息素组分Z9,E11-14:OAc有微弱反应;S1ituOR16对斜纹夜蛾的行为拮抗剂Z9-14:OH有最大反应(EC_(50)=2.920×10~(-6)M);S1ituOR11对所有检测气味均不反应。研究结果为明确斜纹夜蛾性信息素的感受机制提供了重要依据。2.斜纹夜蛾普通气味受体基因的克隆、表达定位及功能研究蛾类受体功能的研究大多针对PR,对non-PR OR的研究很少。我们根据已报道的海灰翅夜蛾的OR序列,设计特异性引物从斜纹夜蛾触角中克隆得到4条non-PR OR片段,进一步通过RACE技术获得cDNA全长序列,分别命名为SlituOR12(Genbank登录号:JX999588)、SlituOR19(JX999589)、SlituOR44(JX999587)和SlituOR51(JX999586)。组织表达谱分析表明,这4条OR都在触角中表达;原位杂交(in situ hybridization)实验表明,SlituOR12在长毛形、短毛形和锥形感器中都有表达,与感器的嗅觉功能相一致。进一步利用非洲爪蟾卵母细胞表达并分析4个OR的功能,结果表明,SlituOR12特异性地识别植物挥发物顺-3-己烯基乙酸酯(EC_(50)=3.393×10-7M),SlituOR19对4'-乙基苯乙酮有微弱反应,但SlituOR44和SlituOR51对所有检测气味均无反应。基于前人对顺-3-己烯基乙酸酯对昆虫行为影响的报道,我们推测SlituOR12参与了雌虫寻找寄主和产卵场所的过程。3.斜纹夜蛾感觉神经元膜蛋白基因的克隆及表达定位感觉神经元膜蛋白是一种膜蛋白,在果蝇中已被证实在性信息素的感受中必不可少。我们利用PCR技术从斜纹夜蛾触角中克隆鉴定了2个SNMP基因的全长cDNA序列,分别命名为SlituSNMP1(GenBank登录号:KC571258)和SlituSNMP2(KC571259)。将2个SNMP进行进化分析发现,两者分别属于SNMP1和SNMP2亚族。利用RT-PCR和qPCR进行组织表达谱分析,发现SlituSNMP1特异表达于成虫触角;而SlituSNMP2主要在成虫触角中表达,在其他嗅觉器官(如喙和下唇须)和非嗅觉器官(足和生殖器)中也有少量表达。利用原位杂交技术定位了这2个基因在雄虫触角中的感器分布,发现SlituSNMP1在长毛形感器下的神经元中表达,而SlituSNMP2则在周围的柱状细胞中表达,暗示两者均参与了嗅觉过程,但功能上有所差异。4.棉铃虫触角转录组测序及嗅觉感受基因的鉴定为了更好地研究棉铃虫的嗅觉识别机制,我们利用Illumina HiSeqTM 2000高通量测序平台对棉铃虫触角进行转录组测序,新发现了15个OR、7个IR、8个OBP和6个CSP。包括本实验室已报道的棉铃虫454测序结果,我们在棉铃虫触角中共鉴定到133个嗅觉基因,包括60个OR、19个IR、2个SNMP、34个OBP和18CSP。这些基因的鉴定为全面阐释棉铃虫的嗅觉机制奠定了重要基础,同时为进一步通过与姐妹种烟青虫进行比较分析来揭示两种昆虫食性分化的机制提供了重要依据。5.棉铃虫气味受体基因的表达谱及功能分析在对棉铃虫进行转录组测序及OR鉴定的基础上,进一步对这些OR进行了功能研究。首先利用RT-PCR技术对已鉴定的所有OR进行了组织表达谱分析,结果显示,其中28个OR基因在成虫触角和幼虫触角中均有表达,2个为幼虫特异表达,其余只在成虫触角表达。然后利用RACE技术克隆了26个OR基因的全长cDNA序列,并对其中的12个OR进行了功能鉴定,对84种气味物质的电生理反应测定发现,7个OR(OR7、8、23、26、27、34和43)的气味结合谱很窄,只对2-4种气味物质有反应;4个OR(OR31、35、40和42)的结合谱较宽,对5种以上气味有反应;1个对被测气味物质均未有反应。推测这些反应谱窄的OR在棉铃虫寄主的识别过程中发挥重要作用。综上所述,本文综合运用分子生物学、生物信息学和电生理记录学技术,在斜纹夜蛾触角中克隆了8个OR(4个PR和4个non-PR OR)和2个SNMP,测定了这些基因的组织表达特征,并进一步鉴定了它们的功能;在棉铃虫中新鉴定了36个嗅觉基因,包括15个OR、7个IR、8个OBP和6个CSP,克隆了26个OR的全长cDNA序列,并完成了12个OR的功能鉴定。研究结果为阐明斜纹夜蛾、棉铃虫对性信息素和植物挥发物的嗅觉分子机制提供了重要依据,同时为设计和开发高效的蛾类行为引诱剂和交配干扰剂提供了支撑。
[Abstract]:As the largest species and number of organisms on earth, insects have developed a highly sensitive olfactory receptor system during their long evolutionary process to sense complex odor compounds in the environment, thus completing important behavioral activities such as spouse-seeking, food-spawning, and escape from natural enemies. From the point of view of application, the long-term use of chemical pesticides in pest control in China has not only resulted in resistance to pests and reduced control effect, but also caused serious potential safety hazards to the environment and human life and caused special attention. It is urgent to develop more environmentally friendly and safer pest control techniques. Sexual attractants based on highly specific pheromone communication between the sexes of insects have been successfully applied to the control of some pests, but the overall effect is still not as expected, mainly because the olfactory mechanism of insect sex pheromones or other odorants is not well understood. Recognition involves multiple proteins, such as odor binding protein (OBP), chemosensory protein (CSP), odor receptor (OR), ionic receptor (IR), sensory neuron membrane protein (SNMP) and odor degrading enzyme (Odorant degrading enzym). Further study on the functions of these genes and their mechanisms in odor recognition will undoubtedly contribute to the development of more efficient pest control techniques. This study focused on the important agricultural pests Spodoptera litura and Helicoverpa armigera, combining molecular biology, bioinformatics and electricity. The main results are as follows: 1. Cloning, expression and function study of sex pheromone receptor gene of Spodoptera litura. According to the functional differences, OR can be divided into sex pheromone receptor (PR) and non-sex pheromone receptor (non-p receptor). Hermone receptor (non-PR OR). According to the four PR genes reported by Spodoptera littoralis, four PR gene fragments were cloned and identified from the antennae of male Spodoptera littoralis, and their full-length cDNA sequences were obtained by RACE, named SlituOR6 (Genbank login number: KC188666), SlituOR11 (KC1). The expression profiles of SlituOR13 (KC188668) and Slituor16 (KC188669) showed that four PRs were mainly expressed in antennae, SlituOR6 and Slituor13 were specifically expressed in antennae of male worms, and SlituOR11 and Slituor16 were highly expressed in antennae of male worms. The specific reactions of tuOR6 to secondary pheromone components Z9, E12-14:OAc (ECu (50) = = 1.99 *10 ~ (-6) M); SlituOR13 to secondary pheromone components Z9, E12-14:OAc (EC_ (50) = = 6.109 *10 ~ (-6) M) and Z9-14:OAc (EC_ (50) = = 1.109 (50) =1.184 10 ~ (-6) M) M) were relatively strong, but slight reactto major phercomponents Z9, E11 1 1 1 1 1 1 1 1 1 1-14:14:OAc (14:OA1: OA (50) =6: OA (50) =6: OA (EC_moth line The results provide an important basis for understanding the sensory mechanism of sex pheromone in Spodoptera litura. 2. Cloning, expression, localization and function of the common odor receptor gene of Spodoptera litura. We cloned four non-PR OR fragments from the antennae of Spodoptera litura by designing specific primers according to the reported OR sequence, and then obtained the full-length cDNA sequences by RACE, named SlituOR12 (Genbank login: JX999588), SlituOR19 (JX999589), SlituOR44 (JX99958). 7) and SlituOR51 (JX999586). Tissue expression profiles showed that the four ORs were expressed in the antennae. In situ hybridization experiments showed that SlituOR12 was expressed in the long hairy, short hairy and pyramidal sensilla, consistent with the olfactory function of sensilla. The results showed that SlituOR12 specifically recognized the plant volatile cis-3-hexenyl acetate (EC_ (50) = 3.393 *10-7M), SlituOR19 had a weak response to 4'-ethylacetophenone, but SlituOR44 and Slituor51 did not respond to all the detected odors. 3. Cloning and localization of the membrane protein gene of the sensory neurons of Spodoptera litura is a membrane protein, which has been proved to be essential in the sensory pheromone of Drosophila melanogaster. We cloned and identified two SNMP bases from the antennae of Spodoptera litura by PCR. The two SNMPs were named SlituSNMP1 (GenBank login number: KC571258) and SlituSNMP2 (KC571259). Evolutionary analysis showed that the two SNMPs belonged to SNMP1 and SNMP2 subgroups, respectively. In situ hybridization was used to localize the distribution of the two genes in the sensilla of male antennae. SlituSNMP1 was expressed in the neurons under the trichomes, while SlituSNMP2 was expressed in the surrounding columnar cells. In order to better understand the olfactory recognition mechanism of Helicoverpa armigera, we used Illumina HiSeqTM 2000 high-throughput sequencing platform to sequence the cotton bollworm antennae, and found 15 OR, 7 IR, 8. A total of 133 olfactory genes, including 60 OR, 19 IR, 2 SNMP, 34 OBP and 18 CSP, were identified in the antennae of Helicoverpa armigera, including 454 sequencing results reported in our laboratory. The identification of these genes laid an important foundation for the comprehensive elucidation of the olfactory mechanism of Helicoverpa armigera, and for the further adoption of sister species of tobacco. The comparative analysis of caterpillars provided important basis for revealing the mechanism of feeding differentiation between the two insects. 5. The expression profiles and functional analysis of odor receptor genes in Helicoverpa armigera were carried out on the basis of transcriptome sequencing and OR identification. The functions of these ORs were further studied. Firstly, all identified ORs were identified by RT-PCR. The results of tissue expression profiles showed that 28 OR genes were expressed in the antennae and antennae of adult worms, 2 were larvae-specific, and the rest were only expressed in the antennae of adult worms. Physical reactions showed that seven ORs (OR7, 8, 23, 26, 27, 34 and 43) had very narrow odor binding spectra and reacted only to 2-4 odor compounds; four ORs (OR31, 35, 40 and 42) had wide binding spectra and reacted to more than five odors; one did not react to the detected odor compounds. To sum up, eight OR (four PR and four non-PR OR) and two SNMPs were cloned from the antennae of Spodoptera litura by using molecular biology, bioinformatics and electrophysiological recording techniques. The tissue expression characteristics of these genes were determined and their functions were further identified. The results provide important basis for elucidating the olfactory molecular mechanism of sex pheromones and plant volatiles in Spodoptera litura and Helicoverpa armigera, and for designing and developing effective behavioral attractants and mating interference in moths. The agent provides support.
【学位授予单位】：南京农业大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：S433.4

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