【作者】 杨微；

【导师】 张泽；

【作者基本信息】 重庆大学 ， 生物学， 2011， 硕士

【摘要】 羧酸酯酶是一种广泛存在于动物,植物及细菌等生物的多功能酶系。根据序列相似性和底物特异性,羧酸酯酶可以分为8个亚家族:α-酯酶,β-酯酶,保幼激素酯酶,乙酰胆碱酯酶,神经趋化蛋白,神经连接蛋白,gliotactin和glutactin。羧酸酯酶家族的基因功能高度分化,部分具有水解活性的α-酯酶或β-酯酶基因在昆虫触角中表达,具有降解性信息素和其他外源气味分子的功能,是昆虫体内重要的气味降解酶。近年来,昆虫触角羧酸酯酶对信息素和植物挥发性气味物质的降解研究使我们对羧酸酯酶基因功能有了新的认识。由于其在嗅觉系统中表达,并具有气味降解活性,因此它们参与了昆虫的嗅觉反应,在维持嗅觉的正常生理功能方面具有至关重要的作用。但是已有的其他研究,仅关注的是昆虫成虫触角中的羧酸酯酶的气味降解功能,但是幼虫的嗅觉组织中是否也存在羧酸酯酶基因的表达,它们能否对外源气味物质进行降解?这一问题值得进一步的研究。家蚕是鳞翅目昆虫的重要模式生物之一,在遗传学研究上具有重要地位。与果蝇、按蚊幼虫不同,家蚕幼虫生活在开放的陆地环境中,是一种寡食性的昆虫,其寄主植物气味信号浓度较低且易受到有毒物质如杀虫剂的影响。研究家蚕COEs幼虫嗅觉组织中羧酸酯酶对挥发性的寄主植物气味物质和杀虫剂的降解作用,可为其他鳞翅目害虫的防治奠定基础。本研究室前期研究发现,家蚕幼虫触角和下颚中至少存在11个羧酸酯酶基因的表达,其中部分基因与已鉴定的鳞翅目昆虫气味降解酯酶和信息素降解酯酶为直系同源基因。本文选取了在家蚕幼虫嗅觉感器表达的Bmae32,Bmae33,Bmae35羧酸酯酶基因,参照ESTs拼接结果设计了特异性引物,从家蚕头组织中克隆这3个基因,并利用生物信息学方法对这些基因编码的蛋白质序列进行预测和分析;利用半定量RT-PCR技术对这3个基因的组织表达模式进行分析;构建Bmae32和Bmae35的原核表达载体,并对Bmae35进行了外源诱导表达,表达产物经镍亲和层析柱纯化,Western blotting鉴定。结果如下:1.经cDNA克隆,家蚕羧酸酯酶Bmae32、Bmae33和Bmae35基因的完整编码序列长度分别为1623 bp、1656 bp和1581 bp,这3个基因均由3个外显子与2个内含子组成,外显子/内含子边界处均符合GT-AG规则。Bmae32基因共分别编码540个氨基酸,其分子量为61.7kD,等电点为8.72,含有1个潜在的N-糖基化位点,19个磷酸化位点;Bmae33基因共分别编码551个氨基酸,其蛋白质分子量为62.15kD,等电点为5.87,含有7个潜在的N-糖基化位点,27个磷酸化位点;Bmae35基因共分别编码526个氨基酸,蛋白质分子量为59.19 kDa,等电点为6.39,含有5个潜在的N-糖基化位点,18个磷酸化位点。2.经Signal IP 3.0在线预测发现Bmae32,Bmae33和Bmae35可能分别含有24、24和15个氨基酸的信号肽,因此这三个蛋白可能均为分泌蛋白。与其他昆虫触角酯酶的多序列比对分析发现Bmae32,Bmae33和Bmae35编码的蛋白具有酯酶活性必须的催化残基:Ser,Glu和His,也保持着α-酯酶家族特征基序Gly-x-Ser-x-Gly。进化分析和序列相似性显示:家蚕这三个羧酸酯酶基因都属于α-酯酶家族,其中Bmae32基因与甘蓝夜蛾触角酯酶基因Mbra-EST可能为直系同源基因,氨基酸一致性为58.8%;Bmae33基因与多音天蚕气味降解酶Apol-ODE和海灰翅夜蛾触角酯酶Slit-EST可能为直系同源基因,与Apol-ODE氨基酸一致性为73.1%,与Slit-EST氨基酸一致性为64.6%;Bmae35与蛀茎夜蛾触角酯酶Snon-EST可能为直系同源基因,氨基酸序列一致性为48.6 %。3.表达模式分析表明:Bmae32基因在家蚕5龄第3天头,血液和丝腺组织中有高表达。Bmae33基因在头,马氏管和体壁组织中有高表达,而在Bmae35在各组织中均有表达,其中在头,脂肪体,马氏管,体壁和丝腺的有高表达。并且Bmae35在雌蛾信息腺中表达,与性信息素合成呈正相关,暗示该基因在信息素合成中起重要作用。4.在去除信号肽和分析酶切位点后重新设计引物克隆,以pET28(a)为表达载体,构建Bmae32和Bmae35重组表达质粒,并测序验证。将测序正确的重组质粒Bmae35/pET28(a)转化至大肠杆菌BL21(DE3)感受态细胞中,经IPTG诱导,SDS-PAGE电泳检测发现该基因以包涵体形式表达,并以镍亲合层析柱纯化,Western blotting鉴定证实Bmae35基因在大肠杆菌中正确表达并得以纯化。更多还原

【Abstract】 Carboxylesterases (COEs) are a multi-function enzymes that occur in animals, plants, and microbes. Based on sequence similarity and substrate specificity, insect COE genes can be subdivided into eight subfamilies:α-esterase,β-esterase, juvenile hormone esterase, acetylcholinesterase, neurotactin, neuroligin, gliotactin and glutactin class. Carboxylesterases have a broad range of functions, some of carboxylesterases belonging toα/β-esterase were expressed in insect antennae, these COEs are important odorant-degrading enzymes that eliminate pheromones and allelochemiacls.Recently, the studies on antennae esterases as a crucial role in inactivating pheromones and plant allelochemicals provided us with useful information for understanding the new functions of these enzymes. Antennae esterases are localized in insect olfaction sensilla and able to degrade odorants compounds. This suggests that they participate in the sense of smell reaction, and play important roles in maintaining the normal physiology reaction. Previous studies mainly focused on the function of insect antanne esterase, which can degrade odorants compounds. However, if the insect larval COEs are also expressed in olfaction tissue, and if they can hydrolyze endogenous, all those questions are still unclear. Bombyx mori is the model organism of Lepidopteran insects and play important role in studying genetics. Silkworm larva is different from Drosophila melanogaster and Anopheles gambiae. Silkworm is phytophagous insect and grows well on mulberry leaves, which will encounter a low chemical signals from its host plant and toxic allelochemicals, such as insecticide. Thus, study on silkworm COEs involved in detoxifying metabolism and olfactory detection will provide useful information for preventing Lepidopteran pests.Previous research in our laboratory suggested that more than 11 COEs were expressed in silkworm larval antenna and maxilla, and some of them are the putative orthologs of insect odorant-degrading esterases and pheromone-degrading esterases. In present study, we cloned three carboxylesterases gene Bmae32, Bmae33 and Bmae35, which were expressed in larval olfactory tissue in the silkworm. According to EST assembling results, we designed primers and cloned those sequence from silkworm head. Then using bioinformatics methods we presumed and analyzed these protein sequences. Based on the semi-quantitative RT-PCR, we determined tissue expression patterns of these genes. At last, The Bmae32 and Bmae35 genes were sub-cloned into the prokaryotic expression vectors. The Bmae35 recombinant protein was expressed in Escherichia coli, purified by immobilized Ni2+ absorption chromatograph column, and proved by Western blotting. All the results are as follows:1. The CDS length of Bmae32, Bmae33, Bmae35 is 1623bp, 1656bp and 1581bp, respectively. The three genes are all composed of 3 exons and 2 introns, and all the boundaries between exons and introns follow the GT-AG rule. Bmae32 encodes 540 amino acids, its molecular mass and isoelectric point are 61.7 kD and 8.72, respectively. It potentially comprises 1 N-glycosylation site and 19 phosphorylation sites. Bmae33 encodes 551 amino acids, its molecular mass and isoelectric point are 62.15 kD and 5.87, respectively. It potentially comprises 7 N-glycosylation site and 27 phosphorylation sites. Bmae35 encodes 526 amino acids, its molecular mass and isoelectric point are 59.19 kD and 6.39, respectively. It potentially comprises 5 N-glycosylation site and 18 phosphorylation sites.2. Signal IP 3.0 analysis revealed that Bmae32, Bmae33 and Bmae35 probably comprise signal peptide with 24, 24 and 15 amino acids long, respectively. Alignment of the Bmae32, Bmae33 and Bmae35 protein sequences with other esterases revealed that all of them have the structure characteristics of insect esterases, such as the catalytic triad (Ser, Glu and His) and the conserved pentapeptide Gly-x-Ser-x-Gly in theα-esterase family. Phylogenetic tree and sequence similarity analysis results indicated that the three COEs belong toα-esterase family, Bmae32 is an putative ortholog of Mbra-EST in Mamestra brassicae with 58.8% identity, Bmae33 is an putative ortholog of Apol-EST in Antheraea polyphemus and Slit-EST in Spodoptera littoralis, with 73.1% and 64.6% identities, respectively, Bmae35 is the putative ortholog of antennal esterase Snon-EST in Sesamia nonagrioide with 48.6% identity.3. The expression pattern showed that Bmae32 was highly expressed in head, hemocyte, and silk gland in day 3 of the fifth instar. Bmae33 was highly expressed in malpighian tubule, epidermis and head. Bmae35 gene was highly expressed in head, fat body, malpighian tubule, epidermis, and silk gland. In addition, the expression of Bmae35 showed positive correlation with sex pheromone content. It suggested that Bmae35 might play important role in pheromone synthesizing.4. We cloned Bmae32 and Bmae35 genes again after removed the signal peptide and analyzed the restriction enzyme site, then constructed the recombined plasmid with pET28(a)vector. The correct recombinant plasmid Bmae35/pET28(a) was transferred into Escherichia coli BL21 (DE3). The recombinant protein was obtained by isopropyl β-D-1-thiogalactopyranoside (IPTG) inducement. Electrophoresis analysis showed that Bmae35 was expressed in Escherichia coli as inclusion body. The recombinant protein Bmae35 was purified by immobilized Ni2+ absorption chromatograph column. Western blotting analysis suggested that Bmae35 was correctly expressed in E. coli and purified.更多还原