【摘要】：作為地球上種類和數(shù)量最大的生物類群,昆蟲在漫長的進化過程中,形成了高度靈敏的嗅覺感受系統(tǒng),以此來感受環(huán)境中紛繁復(fù)雜的氣味物質(zhì),從而完成尋找配偶、食物、產(chǎn)卵場所和逃避天敵等重要的行為活動,因此研究昆蟲嗅覺感受機制具有重要的科學(xué)意義。從應(yīng)用的角度講,我國長期以來采用化學(xué)農(nóng)藥防治害蟲,不僅使害蟲產(chǎn)生了抗性,防治效果降低,同時農(nóng)藥殘留給環(huán)境及人類生活帶來的安全隱患日益嚴(yán)重并引起格外關(guān)注,亟需開發(fā)更環(huán)保、更安全的害蟲防治技術(shù)�；诶ハx兩性之間高度特異的信息素通訊而設(shè)計開發(fā)的性引誘劑已經(jīng)成功應(yīng)用于一些害蟲的防治,但總體效果仍無法達到人們預(yù)期,主要原因是對昆蟲性信息素或其他氣味物質(zhì)的嗅覺機制還不夠了解。昆蟲對于氣味物質(zhì)的感受和識別涉及多個蛋白,如氣味結(jié)合蛋白(Odorant binding protein,OBP)、化學(xué)感受蛋白(Chemosensory protein,CSP)、氣味受體(Odorant receptor,OR)、離子型受體(Ionotropic receptor,IR)、感覺神經(jīng)元膜蛋白(Sensory neuron membrane protein,SNMP)和氣味降解酶(Odorant degrading enzyme,ODE)等。深入研究這些基因的功能及其在氣味識別過程中的作用機制,無疑將有助于開發(fā)更高效的害蟲防治技術(shù)。本研究以重要農(nóng)業(yè)害蟲斜紋夜蛾(Spodoptera litura)和棉鈴蟲(Helicoverpa armigera)為研究對象,結(jié)合分子生物學(xué)、生物信息學(xué)和電生理學(xué)技術(shù),重點對OR和SNMP兩類基因進行了鑒定及功能分析,主要結(jié)果如下:1.斜紋夜蛾性信息素受體基因的克隆、表達及功能研究在鱗翅目昆蟲中,根據(jù)功能差異一般可以將OR分為性信息素受體(pheromone receptor,PR)和非性信息素受體(non-pheromone receptor,non-PR OR)。根據(jù)海灰翅夜蛾(Spodoptera littoralis)報道的4個PR基因,設(shè)計特異性引物,從斜紋夜蛾雄蛾觸角中克隆并鑒定了4個PR基因片段,進一步通過RACE技術(shù)獲得其全長cDNA序列,分別命名為SlituOR6(Genbank登錄號:KC188666)、SlituOR11(KC188667)、SlituOR13(KC188668)和SlituOR16(KC188669)。組織表達譜顯示,4個PR主要在觸角中表達,SlituOR6和SlituOR13在雄蟲觸角特異表達,SlituOR11和SlituOR16在雄蟲觸角高表達。進一步采用非洲爪蟾卵母細胞(Xenopus oocytes)系統(tǒng)表達并分析4個PRs的功能,其中SlituOR6對次要性信息素組分Z9,E12-14:OAc特異反應(yīng)(EC_(50)=1.99×10~(-6) M);SlituOR13對次要性信息素組分Z9,E12-14:OAc(EC_(50)=6.109×10~(-6)M)和Z9-14:OAc(EC_(50)=1.184×10~(-6)M)反應(yīng)比較強烈,對主要性信息素組分Z9,E11-14:OAc有微弱反應(yīng);S1ituOR16對斜紋夜蛾的行為拮抗劑Z9-14:OH有最大反應(yīng)(EC_(50)=2.920×10~(-6)M);S1ituOR11對所有檢測氣味均不反應(yīng)。研究結(jié)果為明確斜紋夜蛾性信息素的感受機制提供了重要依據(jù)。2.斜紋夜蛾普通氣味受體基因的克隆、表達定位及功能研究蛾類受體功能的研究大多針對PR,對non-PR OR的研究很少。我們根據(jù)已報道的海灰翅夜蛾的OR序列,設(shè)計特異性引物從斜紋夜蛾觸角中克隆得到4條non-PR OR片段,進一步通過RACE技術(shù)獲得cDNA全長序列,分別命名為SlituOR12(Genbank登錄號:JX999588)、SlituOR19(JX999589)、SlituOR44(JX999587)和SlituOR51(JX999586)。組織表達譜分析表明,這4條OR都在觸角中表達;原位雜交(in situ hybridization)實驗表明,SlituOR12在長毛形、短毛形和錐形感器中都有表達,與感器的嗅覺功能相一致。進一步利用非洲爪蟾卵母細胞表達并分析4個OR的功能,結(jié)果表明,SlituOR12特異性地識別植物揮發(fā)物順-3-己烯基乙酸酯(EC_(50)=3.393×10-7M),SlituOR19對4'-乙基苯乙酮有微弱反應(yīng),但SlituOR44和SlituOR51對所有檢測氣味均無反應(yīng)�；谇叭藢�-3-己烯基乙酸酯對昆蟲行為影響的報道,我們推測SlituOR12參與了雌蟲尋找寄主和產(chǎn)卵場所的過程。3.斜紋夜蛾感覺神經(jīng)元膜蛋白基因的克隆及表達定位感覺神經(jīng)元膜蛋白是一種膜蛋白,在果蠅中已被證實在性信息素的感受中必不可少。我們利用PCR技術(shù)從斜紋夜蛾觸角中克隆鑒定了2個SNMP基因的全長cDNA序列,分別命名為SlituSNMP1(GenBank登錄號:KC571258)和SlituSNMP2(KC571259)。將2個SNMP進行進化分析發(fā)現(xiàn),兩者分別屬于SNMP1和SNMP2亞族。利用RT-PCR和qPCR進行組織表達譜分析,發(fā)現(xiàn)SlituSNMP1特異表達于成蟲觸角;而SlituSNMP2主要在成蟲觸角中表達,在其他嗅覺器官(如喙和下唇須)和非嗅覺器官(足和生殖器)中也有少量表達。利用原位雜交技術(shù)定位了這2個基因在雄蟲觸角中的感器分布,發(fā)現(xiàn)SlituSNMP1在長毛形感器下的神經(jīng)元中表達,而SlituSNMP2則在周圍的柱狀細胞中表達,暗示兩者均參與了嗅覺過程,但功能上有所差異。4.棉鈴蟲觸角轉(zhuǎn)錄組測序及嗅覺感受基因的鑒定為了更好地研究棉鈴蟲的嗅覺識別機制,我們利用Illumina HiSeqTM 2000高通量測序平臺對棉鈴蟲觸角進行轉(zhuǎn)錄組測序,新發(fā)現(xiàn)了15個OR、7個IR、8個OBP和6個CSP。包括本實驗室已報道的棉鈴蟲454測序結(jié)果,我們在棉鈴蟲觸角中共鑒定到133個嗅覺基因,包括60個OR、19個IR、2個SNMP、34個OBP和18CSP。這些基因的鑒定為全面闡釋棉鈴蟲的嗅覺機制奠定了重要基礎(chǔ),同時為進一步通過與姐妹種煙青蟲進行比較分析來揭示兩種昆蟲食性分化的機制提供了重要依據(jù)。5.棉鈴蟲氣味受體基因的表達譜及功能分析在對棉鈴蟲進行轉(zhuǎn)錄組測序及OR鑒定的基礎(chǔ)上,進一步對這些OR進行了功能研究。首先利用RT-PCR技術(shù)對已鑒定的所有OR進行了組織表達譜分析,結(jié)果顯示,其中28個OR基因在成蟲觸角和幼蟲觸角中均有表達,2個為幼蟲特異表達,其余只在成蟲觸角表達。然后利用RACE技術(shù)克隆了26個OR基因的全長cDNA序列,并對其中的12個OR進行了功能鑒定,對84種氣味物質(zhì)的電生理反應(yīng)測定發(fā)現(xiàn),7個OR(OR7、8、23、26、27、34和43)的氣味結(jié)合譜很窄,只對2-4種氣味物質(zhì)有反應(yīng);4個OR(OR31、35、40和42)的結(jié)合譜較寬,對5種以上氣味有反應(yīng);1個對被測氣味物質(zhì)均未有反應(yīng)。推測這些反應(yīng)譜窄的OR在棉鈴蟲寄主的識別過程中發(fā)揮重要作用。綜上所述,本文綜合運用分子生物學(xué)、生物信息學(xué)和電生理記錄學(xué)技術(shù),在斜紋夜蛾觸角中克隆了8個OR(4個PR和4個non-PR OR)和2個SNMP,測定了這些基因的組織表達特征,并進一步鑒定了它們的功能;在棉鈴蟲中新鑒定了36個嗅覺基因,包括15個OR、7個IR、8個OBP和6個CSP,克隆了26個OR的全長cDNA序列,并完成了12個OR的功能鑒定。研究結(jié)果為闡明斜紋夜蛾、棉鈴蟲對性信息素和植物揮發(fā)物的嗅覺分子機制提供了重要依據(jù),同時為設(shè)計和開發(fā)高效的蛾類行為引誘劑和交配干擾劑提供了支撐。
[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.
【學(xué)位授予單位】：南京農(nóng)業(yè)大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2015
【分類號】：S433.4

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10 許冬;斜紋夜蛾取食誘導(dǎo)棉花抑制性消減文庫的構(gòu)建及初步分析[D];中國農(nóng)業(yè)科學(xué)院;2008年

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