绿盲蝽论文绿盲蝽气味结合蛋白的分子识别功能

1、绿盲蜻论文：绿盲蜻气味结合蛋白的分子识别功能【中文摘要】嗅觉在昆虫多种行为方面起着重要的作用，比如， 农业害虫的寄主选择行为、择偶行为等等。昆虫的气味结合蛋白(obps)是一类存在于嗅觉感受器淋巴液中的可溶性的分泌蛋白，它 能运输气味分了到达嗅觉神经表面的受体上，是昆虫对外界气味分了 的识别和化学信息素传递过程中的第一步生化反应。利用昆虫的嗅觉 识别机制，以obps为靶标基因，干扰昆虫的寄主定位与交配等行为, 将这一研究用于农业牛产中的害虫治理已经成为近年来研究的热点， 具有一定的理论与实践意义。绿盲蜻是一种重要的、杂食性的农业昆 虫，是我国长江流域与黄河流域棉田害虫的优势种，它不仅可以为害

2、棉花、苜蓿、枣树、向日葵、茴蒿等，同时多种果树及蔬菜都是绿盲 蜻的寄主。本研究以绿盲plus-cllucobpl为研究目标，对其进行 了原核表达，并对其进行了功能验证。本研究的主要研究结果有以下 几点：1选择出了适合plus-c气味结合蛋白的表达菌株，经过稀有 密码子分析与一系列的预实验，最终选择rosetta(de3)为plus-c obp1气味结合蛋白的表达菌株，避免了稀有密码子对蛋白表达的影 响。2.克隆并表达了第一个绿盲嬉plus-c气味结合蛋白-alucobpl, 通过质谱鉴定与western blot验证了表达蛋白，确定其带标签分子 量在37kd左右，运用histrap亲和层析柱法

3、纯化蛋白，发现300mm 咪啜是洗脱的最适浓度。切标签后发现其分子量为25kd左右，运用 bradford法测得最终纯化蛋白的浓度为1. 462 mg/mlc 3.以1-npn为 荧光探针，通过荧光竞争结合实验，检测了 50种不同种类的包括植 物挥发物及性信息素的气味分子与alucobpl的结合能力，结果显示, alucobpl能与多种气味分子结合，其中与十七烷醇、1-庚醇、丁酸 己酯、丁酸丁酯、金合欢烯、罗勒烯、2-辛酮等几种气味分子的结合 能力比较强。推测alucobpl可能在感受信息素以及寄主定位的过程 中发挥着重要作用。【英文摘要】olfaction plays a crucial r

4、ole in many kinds of insect behaviors, including host selection and mating behavior. insect odorant-bindingprotein (obp) is one kind of soluble secreted protein existing in thelymph of the olfactory receptor which is associated with olfaction. this protein can transport, as vector, the odour molecul

5、e to thereceptors on the surface of nerve cel1. obp is thought to be the firststep in molecular recognition and the transportation of semi-chemica 1. and it has become a hot spot of research in recent years in many ofthese studies, obps were the target genes, recognition mechanism ofinsect smell sen

6、se was applied, interferenee with insect, s matingand host positioning behaviors that has much theoretical andpractical application are the focus for these researchesaygus 1ucorum, one species of major phytophagous agricultural insect pests that not only cause huge losses in crops like cotton, alfal

7、fa, sunflower etc， but also to a variety of fruit trees (like chinesedate) and vegetables (like artemisia) in this study, aygus lucorumplus-c obp was expressed in a prokaryotic expression system, in turn its function was verified using fluorescence probe technique. mainresults of this study are list

8、ed as the following:1. a suitable expression strain was selected for plus-c obp study .to avoid the rare anticodon influencing the protoin expression, ascrics of rare anticodon analysis were conducted; and in the end, rosetta (de3) was selected as the expression strain of llucobpl. 2. a novel plus-c

9、 obp (alucobpl) in the aygus lucorum wasexpressed and determined a novel plus-c obp (alucobpl) in theaygus lucorum, after being cioned, expressed and purified, and then itwas verified to be alucobpl by mass spectrometry appraisal andwestern blot, that determined its molecular weight about 37 kd byhi

10、stag after purification with histrap affinity chromatography, itwas found that both 200mm and 300mm were its optimal elutionconcentrations its molecular weight would become about 25 kd aftercutting down ilistag its protein concentration was 1.462 mg/mlmeasured by bradford method.3. the binding effic

11、iency of alucobpl with 50 odorants of differentchemical classes was measured using a fluorescence probe 1-npn inafluorescence competitive binding study. results showed that manyodorants can bind with alucobpl, among which,heptadecanol, 1-heptanol, ethyl butyrate, nbutyl butyrate, farnesene, ocimene,

12、 2-octdnone could significanlly bind with llucobp 1. these resultssuggested that alucobpl might play a key role in perception of sexpheromones and host selection for this insect species【关键词】绿盲蜡气味结合蛋白plus-c气味结合蛋白原核表 达荧光竞争结合【英文关键词】aygus lucorumodorant-bindingproteinsplus-c obpprokaryotic expressionflu

13、orescence competitive binding【目录】绿盲嬉气味结合蛋白的分子识别功能 英文缩略表6-7 摘要 7-9 abstract 9-101 前言 12-201. 1 绿盲蜻的研究概况12-141. 1. 1绿盲蜻的生物习性12-131. 1.2绿盲蜻的为害特点131. 1.3绿盲蜻的防治13-141.2昆虫的嗅觉行为14-161.2. 1昆虫触角感受器的研究14-151.2.2绿盲蜻的嗅觉感器研究15-161.3昆虫嗅觉的化学感受研究16- 181.3. 1气味结合蛋口的一般特征16-171.3.2气味结合蛋白的作用及功能171.3.3昆虫气味结合蛋白研究进展17- 18

14、1.3.4半翅目昆虫的气味结合蛋白研究进展181.4研究依据及目的和意义18-202绿盲蜻气味结合蛋白的克隆及表达20-362. 1材料与方法202. 1. 1实验材料202. 1. 2试剂202. 1. 3仪器202. 2克隆与表达绿盲蜻气味结合蛋白0bp1 20-282. 2. 1绿盲蜷气味结合蛋白0bp1的克隆20-252. 2. 1. 1样品的准备202. 2. 1. 2绿盲蜡触角rna的提取 20-212. 2. 1. 3 反转录合成第一条 cdna 链 212. 2. 1. 4 pcr扩增目的序列21-222. 2. 1.5目的片段胶回收222. 2. 1. 6 dna与pgem-

15、t载体连接222. 2. 1. 7转化连接产物22-232. 2. 1. 8提取质粒232. 2. 1. 9菌液pcr检测obp1 232. 2. 1. 10用bamhk xho i 酶切 0bp1 23-242.2. 1. 11 用 bamhe xho i 酶切载体pet-30 (a) 242.2. 1. 12密码子分析242. 2. 1. 13目的片段0bp1与表达载体pet-30 (a)连接24-252. 2. 1. 14将连接后的产物转入到感受态细胞dh5 a 252. 2. 1. 15提取表达质粒25 2. 2. 1. 16将表达质粒转化到rosetta(de3)中252. 2.2绿

16、盲蜡气味结合蛋白0bp1的表达25-282. 2. 2. 1诱导表达0bp1 25-262. 2. 2. 2 sds-page 电泳检测 0bp1 的表达情况26 2. 2. 2. 3包涵体的处理262. 2. 2. 4纯化蛋白26-272. 2. 2. 5 western blot 272. 2. 2. 6 质谱分析27 2. 2. 2. 7 透析 27-282. 2. 2. 8 超滤 282. 2. 2. 9 切除ilis-tag 282.2.2. 10 bradford 法测蛋口浓度 282.3 结果与分析28-362. 3. 1绿盲蜻气味结合蛋白0bp1的序列分析28-302. 3. 2绿盲嬉触角rna 302. 3. 3绿盲蜻气味结合蛋白0bp1的克隆30-322. 3. 4稀有密码子分析结果322. 3. 5绿盲嬉气味结合蛋白0bp1的表达32-332. 3. 6质谱结果33-342. 3. 7 western blot 结果 34-352. 3. 8 切