【摘要】：暗黑鰓金龜（Holotrichia parallela Motschulsky）屬于鞘翅目鰓金龜科，其幼蟲(chóng)稱蠐螬，是重要的地下害蟲(chóng)，對(duì)農(nóng)作物、蔬菜和果樹(shù)能引發(fā)嚴(yán)重的為害。因此，有效的防治暗黑鰓金龜已成為亟待解決的問(wèn)題。嗅覺(jué)系統(tǒng)對(duì)于昆蟲(chóng)尋找寄主、配偶等行為至關(guān)重要，了解昆蟲(chóng)嗅覺(jué)系統(tǒng)將有助于研發(fā)出新的防治策略來(lái)治理害蟲(chóng)，包括利用昆蟲(chóng)的嗅覺(jué)識(shí)別機(jī)制驅(qū)散或吸引害蟲(chóng)以達(dá)到防治的目的。差異蛋白質(zhì)組學(xué)能夠揭示嗅覺(jué)相關(guān)蛋白，是解析觸角嗅覺(jué)分子識(shí)別機(jī)制的重要措施。為了明確暗黑鰓金龜?shù)挠|角嗅覺(jué)識(shí)別機(jī)制，我們應(yīng)用蛋白質(zhì)雙向電泳(2-DE)、質(zhì)譜鑒定以及生物信息學(xué)分析技術(shù)對(duì)暗黑鰓金龜雌雄觸角蛋白進(jìn)行差異分析和功能驗(yàn)證。 蛋白樣品的制備是2-DE蛋白質(zhì)組學(xué)研究的關(guān)鍵環(huán)節(jié)之一。為了建立一個(gè)穩(wěn)定的觸角蛋白質(zhì)組學(xué)研究平臺(tái)，我們對(duì)四種觸角蛋白的制備方法（TCA/丙酮沉淀法、飽和酚法、PEG法和直接提取法）進(jìn)行了分析和優(yōu)化。根據(jù)蛋白產(chǎn)率、SDS-PAGE電泳以及蛋白點(diǎn)數(shù)的分析發(fā)現(xiàn)，TCA/丙酮沉淀法是提取觸角蛋白的最優(yōu)方法。 應(yīng)用軟件ImageMaster2D Platinum7.0對(duì)銀染后的蛋白質(zhì)雙向電泳圖譜進(jìn)行分析后發(fā)現(xiàn)，每張凝膠上能檢測(cè)到約1100個(gè)蛋白點(diǎn)。差異蛋白分析表明，雌雄觸角里總共有47個(gè)蛋白點(diǎn)發(fā)生了顯著性（1.5fold，p-value 0.05）變化。其中，25個(gè)蛋白點(diǎn)在雄蟲(chóng)觸角里上調(diào)表達(dá)，22個(gè)蛋白點(diǎn)在雌蟲(chóng)觸角里上調(diào)表達(dá)。為了鑒定觸角里的差異表達(dá)蛋白，運(yùn)用MALDI-TOF/TOF MS質(zhì)譜技術(shù)對(duì)從凝膠上分離下來(lái)的蛋白點(diǎn)進(jìn)行分析。47個(gè)差異表達(dá)蛋白中有35個(gè)被成功鑒定。在這些蛋白中，65.7%的蛋白與碳水化合物和能量代謝、抗氧化系統(tǒng)、轉(zhuǎn)運(yùn)以及氨基酸和核苷酸代謝有關(guān)。部分嗅覺(jué)相關(guān)蛋白首次被鑒定，包括肽聚糖識(shí)別蛋白-1（spot16），酰基輔酶A脫氫酶(spot26)，磷酸丙酮異構(gòu)酶類似物(spot29)，醛酮還原酶類似物(spot34)、絲氨酸蛋白酶(spot21)，氣味結(jié)合蛋白2(spot38)，，乙醛脫氫酶(spot14)，細(xì)胞色素P450（spot12）等。 依據(jù)KEGG和Gene Ontology數(shù)據(jù)庫(kù)和軟件對(duì)差異表達(dá)蛋白進(jìn)行生物信息學(xué)分析，將鑒定出的蛋白共分成8類。其中，20.0%的蛋白與碳水化合物和能量代謝相關(guān)，17.1%的蛋白與抗氧化系統(tǒng)相關(guān)，14.3%的蛋白與轉(zhuǎn)運(yùn)相關(guān),14.3%的蛋白與氨基酸和核苷酸代謝相關(guān)。另外還包括11.4%的細(xì)胞骨架蛋白和8.6%與蛋白質(zhì)折疊相關(guān)的蛋白。 對(duì)鑒定的6個(gè)嗅覺(jué)相關(guān)蛋白進(jìn)行了蛋白質(zhì)互作網(wǎng)絡(luò)分析，包括熱激蛋白70同源物-3、絲氨酸蛋白酶和醛酮還原酶類似物，以及觸角里含量豐富的細(xì)胞色素P450、乙醛脫氫酶和谷氨酰胺合成酶，為蛋白質(zhì)功能的進(jìn)一步研究創(chuàng)造了條件。應(yīng)用實(shí)時(shí)熒光定量PCR技術(shù)檢測(cè)了6個(gè)嗅覺(jué)相關(guān)蛋白在轉(zhuǎn)錄水平上的變化，結(jié)果顯示mRNA與蛋白質(zhì)水平的變化基本一致。
[Abstract]:(Holotrichia parallela Motschulsky) belongs to the family Coleoptera, and its larvae are called grub, which is an important underground pest, which can cause serious damage to crops, vegetables and fruit trees. Therefore, the effective prevention and cure dark gills golden tortoise has become the question which urgently needs to be solved. The olfactory system is essential for insects to find hosts, mates and other behaviors. Understanding the insect olfactory system will help to develop new control strategies to control pests. Including the use of insect olfactory recognition mechanism to disperse or attract pests for control purposes. Differential proteomics, which can reveal olfactory related proteins, is an important measure to elucidate the molecular recognition mechanism of antennae olfaction. In order to clarify the mechanism of olfactory recognition of the antennae, we used protein two-dimensional electrophoresis (2-DE), mass spectrometry (MS) and bioinformatics analysis techniques to analyze and verify the function of male and female antennae proteins. The preparation of protein sample is one of the key links in 2-DE proteomics. In order to establish a stable platform for the study of antennal proteomics, four methods for the preparation of antennal proteins (TCA/ acetone precipitation method, saturated phenol method, PEG method and direct extraction method) were analyzed and optimized. According to protein yield, SDS-PAGE electrophoresis and protein number analysis, it was found that TCA/ acetone precipitation method was the best method for extracting antennal proteins. Using software ImageMaster2D Platinum7.0 to analyze the two dimensional electrophoresis patterns of silver-stained proteins, it was found that about 1,100 protein spots could be detected on each gel. The results of differential protein analysis showed that there were significant changes in 47 protein spots (1.5 foldsp-value 0.05) in the male and female antennae. Among them, 25 protein spots were up-regulated in male antennae and 22 protein spots were up-regulated in female antennae. In order to identify differentially expressed proteins in the antennae, the protein spots isolated from the gel were analyzed by MALDI-TOF/TOF MS mass spectrometry. 35 of the 47 differentially expressed proteins were successfully identified. Of these proteins, 65.7% were related to carbohydrate and energy metabolism, antioxidant system, transport and metabolism of amino acids and nucleotides. Some olfactory related proteins have been identified for the first time, including peptidoglycan recognition protein-1 (spot16), acyl-coenzyme A dehydrogenase (spot26), phosphoacetone isomerase analogues (spot29), aldehyde-ketone reductase analogues (spot34), serine protease (spot21), etc. Smell binding protein 2 (spot38), acetaldehyde dehydrogenase (spot14), cytochrome P450 (spot12), etc. According to KEGG and Gene Ontology database and software, the differentially expressed proteins were analyzed by bioinformatics, and the identified proteins were classified into 8 categories. Among them, 20.0% of proteins were related to carbohydrate and energy metabolism, 17.1% to antioxidant system, 14.3% to transport, 14.3% to amino acid and nucleotide metabolism. It also included 11.4% cytoskeleton protein and 8.6% protein related to protein folding. Six olfactory related proteins were identified by protein interaction network analysis, including heat shock protein 70 homologues-3, serine protease and aldehyde-ketone reductase analogues, and cytochrome P450, which is abundant in the antennae. Aldehyde dehydrogenase and glutamine synthase create conditions for further study of protein function. The changes of six olfactory related proteins at the transcriptional level were detected by real-time fluorescence quantitative PCR. The results showed that the changes of mRNA and protein levels were basically the same.
【學(xué)位授予單位】：吉林大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類號(hào)】：S433.5

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