棉铃虫性信息素结合蛋白结构特征及功能分化研究

发布时间：2018-06-03 03:34

本文选题：棉铃虫 + 性信息素结合蛋白　；参考：《中国农业大学》2017年博士论文

【摘要】：棉铃虫Helicoverpaarmigera(Hubner)是一种危害性很大的世界级农业害虫。其与多数种类鳞翅目害虫一样,对同种性信息素有着较强的敏感性。然而时至今日,棉铃虫性信息素识别的分子机制仍有待进一步研究和阐明。性信息素结合蛋白(pheromone binding proteins,PBPs)是一类于昆虫触角感器中广泛存在的水溶性小分子量蛋白,此类蛋白被认为在昆虫的性信息素感受过程中发挥重要作用。为了更好的阐明性信息素结合蛋白在棉铃虫性信息素识别过程中的功能分化及分子机制,本研究采用扫描及透射电镜方法,对棉铃虫触角感器的外形及其内部构造进行了观察,之后利用免疫组化方法对棉铃虫3个已知的PBP蛋白在雌雄蛾触角感器中的表达分布进行定位;同时,本研究还利用原核表达及亲和层析方法,获得了高纯度的棉铃虫性信息素结合蛋白HarmPBP1,并以此为基础筛选得到了该蛋白的单晶及其与配体Z9-16:Ald的复合物晶体结构;以HarmPBP1/Z9-16:Ald的结构为依据,本研究进一步预测了 HarmPBP1与配体结合的可能关键位点,并通过定点突变和荧光竞争结合实验方法对这些位点进行了验证;最后,通过RNAi对棉铃虫HarmPBP1-3基因的功能分化进行研究。主要结论如下:1、棉铃虫雌雄蛾触角均为丝状。在触角上总计观察到6种感器,分别为毛形感器、刺形感器、锥形感器、耳形感器、腔锥形感器和栓锥形感器。毛形感器在所有感器中数量最多,其感器壁较厚,内有1-3个树突神经元;锥形感器壁薄,感器内有大量树突神经元;刺形感器感器壁厚,端部生有顶孔,感器内又分成内外两个淋巴腔,树突神经元分布在内腔中;耳形感器壁薄,感器内有多个树突神经元。2、棉铃虫的3个PBP蛋白在雌雄蛾触角毛形感器淋巴液中均被标记,其中雌蛾触角毛形感器中标记较少;其它感器如刺形感器,锥形感器和腔锥形感器中未发现胶体金标记。3、棉铃虫HarmPBP1蛋白的单晶结构整体由6个α螺旋组成,结合腔主要由疏水氨基酸构成,其整体结构与典型的蛾类PBP结构极为相似;通过HarmPBP1/Z9-16:Ald复合物结构,预测Ser9,Phe12,Ser56和Phe119是HarmPBP1蛋白与配体结合的可能关键位点。4、通过定点突变获得HarmPBP1的5个突变蛋白:F12A、S56A、F119A、S9A和Q64A。荧光竞争结合实验结果显示,除随机突变Q64A外,其余4种突变蛋白与棉铃虫性信息素的两种主要组分Z11-16:Ald和Z9-16:Ald的结合能力与原蛋白相比均出现了明显下降,证明这4个氨基酸残基在HarmPBP1蛋白与配体的结合过程中发挥重要作用。5、对HarmPBP1-3基因进行单独及混合基因干涉,荧光定量结果显示3个基因的表达量被明显抑制;EAG结果显示,3个基因的单独干扰均无法引起雄蛾触角对气味标样的反应下调;当HarmPBP1和HarmPBP2基因被同时干涉后,雄蛾对Z11-16:Ald的EAG反应显著降低;而其他混合干涉雄蛾没有表现出EAG反应下调,表明HarmPBP1和HarmPBP2蛋白可能在雄蛾对Z11-16:Ald的识别过程中共同发挥主要作用。对HarmPBP1和HarmPBP2基因混合干涉雄蛾的风洞试验进一步证实了这个结论。
[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.
【学位授予单位】：中国农业大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：S433.4

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