本文選題：棉鈴蟲(chóng) + 性信息素結(jié)合蛋白��； 參考：《中國(guó)農(nóng)業(yè)大學(xué)》2017年博士論文

【摘要】：棉鈴蟲(chóng)Helicoverpaarmigera(Hubner)是一種危害性很大的世界級(jí)農(nóng)業(yè)害蟲(chóng)。其與多數(shù)種類鱗翅目害蟲(chóng)一樣,對(duì)同種性信息素有著較強(qiáng)的敏感性。然而時(shí)至今日,棉鈴蟲(chóng)性信息素識(shí)別的分子機(jī)制仍有待進(jìn)一步研究和闡明。性信息素結(jié)合蛋白(pheromone binding proteins,PBPs)是一類于昆蟲(chóng)觸角感器中廣泛存在的水溶性小分子量蛋白,此類蛋白被認(rèn)為在昆蟲(chóng)的性信息素感受過(guò)程中發(fā)揮重要作用。為了更好的闡明性信息素結(jié)合蛋白在棉鈴蟲(chóng)性信息素識(shí)別過(guò)程中的功能分化及分子機(jī)制,本研究采用掃描及透射電鏡方法,對(duì)棉鈴蟲(chóng)觸角感器的外形及其內(nèi)部構(gòu)造進(jìn)行了觀察,之后利用免疫組化方法對(duì)棉鈴蟲(chóng)3個(gè)已知的PBP蛋白在雌雄蛾觸角感器中的表達(dá)分布進(jìn)行定位;同時(shí),本研究還利用原核表達(dá)及親和層析方法,獲得了高純度的棉鈴蟲(chóng)性信息素結(jié)合蛋白HarmPBP1,并以此為基礎(chǔ)篩選得到了該蛋白的單晶及其與配體Z9-16:Ald的復(fù)合物晶體結(jié)構(gòu);以HarmPBP1/Z9-16:Ald的結(jié)構(gòu)為依據(jù),本研究進(jìn)一步預(yù)測(cè)了 HarmPBP1與配體結(jié)合的可能關(guān)鍵位點(diǎn),并通過(guò)定點(diǎn)突變和熒光競(jìng)爭(zhēng)結(jié)合實(shí)驗(yàn)方法對(duì)這些位點(diǎn)進(jìn)行了驗(yàn)證;最后,通過(guò)RNAi對(duì)棉鈴蟲(chóng)HarmPBP1-3基因的功能分化進(jìn)行研究。主要結(jié)論如下:1、棉鈴蟲(chóng)雌雄蛾觸角均為絲狀。在觸角上總計(jì)觀察到6種感器,分別為毛形感器、刺形感器、錐形感器、耳形感器、腔錐形感器和栓錐形感器。毛形感器在所有感器中數(shù)量最多,其感器壁較厚,內(nèi)有1-3個(gè)樹(shù)突神經(jīng)元;錐形感器壁薄,感器內(nèi)有大量樹(shù)突神經(jīng)元;刺形感器感器壁厚,端部生有頂孔,感器內(nèi)又分成內(nèi)外兩個(gè)淋巴腔,樹(shù)突神經(jīng)元分布在內(nèi)腔中;耳形感器壁薄,感器內(nèi)有多個(gè)樹(shù)突神經(jīng)元。2、棉鈴蟲(chóng)的3個(gè)PBP蛋白在雌雄蛾觸角毛形感器淋巴液中均被標(biāo)記,其中雌蛾觸角毛形感器中標(biāo)記較少;其它感器如刺形感器,錐形感器和腔錐形感器中未發(fā)現(xiàn)膠體金標(biāo)記。3、棉鈴蟲(chóng)HarmPBP1蛋白的單晶結(jié)構(gòu)整體由6個(gè)α螺旋組成,結(jié)合腔主要由疏水氨基酸構(gòu)成,其整體結(jié)構(gòu)與典型的蛾類PBP結(jié)構(gòu)極為相似;通過(guò)HarmPBP1/Z9-16:Ald復(fù)合物結(jié)構(gòu),預(yù)測(cè)Ser9,Phe12,Ser56和Phe119是HarmPBP1蛋白與配體結(jié)合的可能關(guān)鍵位點(diǎn)。4、通過(guò)定點(diǎn)突變獲得HarmPBP1的5個(gè)突變蛋白:F12A、S56A、F119A、S9A和Q64A。熒光競(jìng)爭(zhēng)結(jié)合實(shí)驗(yàn)結(jié)果顯示,除隨機(jī)突變Q64A外,其余4種突變蛋白與棉鈴蟲(chóng)性信息素的兩種主要組分Z11-16:Ald和Z9-16:Ald的結(jié)合能力與原蛋白相比均出現(xiàn)了明顯下降,證明這4個(gè)氨基酸殘基在HarmPBP1蛋白與配體的結(jié)合過(guò)程中發(fā)揮重要作用。5、對(duì)HarmPBP1-3基因進(jìn)行單獨(dú)及混合基因干涉,熒光定量結(jié)果顯示3個(gè)基因的表達(dá)量被明顯抑制;EAG結(jié)果顯示,3個(gè)基因的單獨(dú)干擾均無(wú)法引起雄蛾觸角對(duì)氣味標(biāo)樣的反應(yīng)下調(diào);當(dāng)HarmPBP1和HarmPBP2基因被同時(shí)干涉后,雄蛾對(duì)Z11-16:Ald的EAG反應(yīng)顯著降低;而其他混合干涉雄蛾沒(méi)有表現(xiàn)出EAG反應(yīng)下調(diào),表明HarmPBP1和HarmPBP2蛋白可能在雄蛾對(duì)Z11-16:Ald的識(shí)別過(guò)程中共同發(fā)揮主要作用。對(duì)HarmPBP1和HarmPBP2基因混合干涉雄蛾的風(fēng)洞試驗(yàn)進(jìn)一步證實(shí)了這個(gè)結(jié)論。
[Abstract]:Helicoverpa armigera Helicoverpaarmigera (Hubner) is a world class agricultural pest with great harm. Like most species of Lepidoptera pests, it has a strong sensitivity to the allogeneic pheromone. However, today, the molecular mechanism of the identification of Helicoverpa armigera is still to be studied and clarified. Pheromon E binding proteins, PBPs) is a kind of small water soluble small molecular weight protein widely existed in insect antennae. This protein is considered to play an important role in the process of insect sex pheromone sensing. In order to better clarify the functional differentiation and molecular mechanism of sex pheromone binding protein in the identification of Helicoverpa armigera sex pheromone, this paper Scanning and transmission electron microscopy were used to observe the shape and internal structure of the antennae of cotton bollworm, and then the expression distribution of 3 known PBP proteins in the antennae of female and male moth was located by immunohistochemistry. At the same time, the study also made use of prokaryotic expression and affinity chromatography. The purity of cotton bollworm sex pheromone binding protein HarmPBP1, based on which the crystal structure of the protein and the crystal structure of the complex with the ligand Z9-16:Ald, is screened. Based on the structure of HarmPBP1/Z9-16:Ald, this study further predicts the key loci of the binding of HarmPBP1 to the ligand, and through the fixed-point mutation and the fluorescence race. Finally, the functional differentiation of HarmPBP1-3 gene of Helicoverpa armigera was studied by RNAi. The main conclusions were as follows: 1, the antennae of the female and male moth of the cotton bollworm were filamentous. A total of 6 sensilla were observed on the antennae, such as hair sensory, cone, ear, and cone-shaped. And taper sensilla. The hair shaped sense organ has the largest number in all sensilla, with the thicker wall of the sensilla and 1-3 dendritic neurons in the sense organ; the cone sense organ has a thin wall and a large number of dendritic neurons in the sensilla; the prickly sensilla is thick, the end has a top hole, and the inner and outer two drenched chambers are in the sensilla, and the dendritic neurons are distributed in the inner cavity; the ear shaped sense organ walls are thin. There are several dendritic neurons.2 in the sensilla, and 3 PBP proteins of the Helicoverpa armigera are marked in the lymph of the antennae hair sense organ of the male and male moths, among which the female moth's tentacle hair sense organ is less marked; the other sensilla, such as the prickly sensilla, the cone sensilla and the cavity cone, are not found in the colloidal gold mark.3, and the single crystal structure of the HarmPBP1 protein of the Helicoverpa armigera is made up of 6. The structure of the binding cavity is mainly composed of hydrophobic amino acids. The overall structure is very similar to the typical PBP structure of the moth. Through the structure of HarmPBP1/Z9-16:Ald complex, it is predicted that Ser9, Phe12, Ser56 and Phe119 are the possible key loci of the binding of HarmPBP1 protein to the ligand, and the 5 mutant proteins of HarmPBP1 are obtained by fixed point mutation: F12A, S5. The experimental results of 6A, F119A, S9A and Q64A. fluorescence competition show that the binding ability of the other 4 mutant proteins to the two major components of Helicoverpa armigera and the Z11-16:Ald and Z9-16:Ald of Helicoverpa armigera is significantly lower than that of the original protein, which proves that the 4 amino acid residues are in the binding process of HarmPBP1 protein with the ligand. .5 was played an important role in the interference of the HarmPBP1-3 gene alone and mixed gene. The fluorescence quantitative results showed that the expression of the 3 genes was obviously suppressed. The EAG results showed that the interference of the male moth's tentacle to the odor standard was not caused by the interference of the male moth's tentacles, and the male moth was to Z11-16:A when the HarmPBP1 and HarmPBP2 genes were simultaneously interfered. The EAG reaction of LD decreased significantly, while other mixed interference male moths did not show a downregulation of EAG reaction, suggesting that HarmPBP1 and HarmPBP2 proteins may play a major role in the identification of Z11-16:Ald by male moths. This conclusion is confirmed by the wind tunnel test of the mixing of HarmPBP1 and HarmPBP2 genes in male moths.
【學(xué)位授予單位】：中國(guó)農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2017
【分類號(hào)】：S433.4

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