【作者】 王东；

【导师】 沈卫德；

【作者基本信息】 苏州大学 ， 特种经济动物饲养学， 2009， 博士

【摘要】 鳞翅目昆虫的许多成员是重要的农林害虫,每年给全世界农林业造成难于估量的损失。由于杀虫剂的广泛使用,许多害虫都产生了不同程度的抗药性,给防治工作带来了极大的困难。细胞色素P450是血红蛋白的超家族,对多样的外源化合物和内源化合物的氧化代谢起作用。在昆虫中,已有大量研究表明细胞色素P450酶系对菊酯类杀虫剂以及植物毒素的解毒代谢方面起着非常重要的作用。羧酸酯酶属于α/β水解酶折叠蛋白,组成一个多基因超家族。它能有效催化含羧基酯键,酰胺键和硫酯键的内源性与外源性多种化合物的水解。在昆虫中,关于羧酸酯酶的研究主要是集中于其在杀虫剂抗性中的作用。此外,已有许多研究表明许多代谢酶类如细胞色素P450和羧酸酯酶都参与了对气味信号分子的降解作用。家蚕是继果蝇和冈比亚按蚊等昆虫之后又一个全基因组测序昆虫,是国际鳞翅目基因组协会确定的鳞翅目昆虫的模式昆虫,而且也是目前唯一的全序列测序鳞翅目昆虫。大量的基因组信息和EST数据库为利用生物信息学方法研究新基因及其功能提供了非常大的便利。家蚕和野桑蚕具有共同的起源,同鳞翅目蚕蛾科昆虫。由于农药对其选择压力的不同,家蚕和野桑蚕对农药的抗性存在差异。对与解毒代谢和嗅觉检测相关的细胞色素P450和羧酸酯酶家族基因的研究,可以为鳞翅目等害虫的防治提供借鉴,同时也为研究这两大家族基因在昆虫中所起的生理功能,以及驯化条件下物种进化差异研究提供参考。本研究利用生物信息学和分子生物学技术从家蚕中鉴定了多个细胞色素P450和羧酸酯酶基因,从分子水平上分析了这些基因的进化关系,研究了基因组织表达特性和发育特性,以及外源毒素对这些基因的诱导表达作用;利用昆虫杆状病毒表达系统表达了部分基因,并对1个羧酸酯酶基因进行了活性鉴定;首次研究比较了家蚕和野桑蚕酯酶在生化和分子水平上的差异,并从农业害虫棉铃虫中克隆了第一个羧酸酯酶基因。1.家蚕细胞色素P450基因的研究本文利用生物信息学和RT-PCR方法从家蚕中克隆了2个CYP4和4个CYP6家族基因序列。序列分析表明这些基因都具有P450基因保守特征位点,而且都在相同位置含有一个保守的内含子/外显子结合位点。CYP4M5和CYP4M9在基因组上以head-to-tail的方式成簇排列,可能是通过基因复制产生的。BLAST结果表明这些P450s与主要参与外源物代谢解毒作用的其他昆虫CYP4和CYP6家族成员同源性较高。RT-PCR分析表明这6个P450基因都在中肠或脂肪体中高表达,基因不同发育阶段表达分析表明它们具有类似或完全不同的表达调控模式。在人工饲料中添加1.75×10-5 %和3.5×10-6 %的氯氰菊酯(cypermethrin),以及0.1%芸香苷(rutin)后,CYP6AB5表达量分别升高了2.3倍,2.2倍和1.9倍;而添食0.1%(槲皮素)quercetin后CYP6AB5表达量基本不变。与此相反,其他5个基因的表达反而都受到不同程度的抑制。我们利用昆虫Bac-to-Bac杆状病毒表达系统表达了CYP6AB5基因。该基因的表达为进一步构建体外代谢酶系,检测该基因的底物特异性打下了基础。信号失活是嗅觉动态过程的一个重要步骤,这一过程包括多样的气味降解酶类。本文利用RT-PCR方法从家蚕蛾的触角中克隆了另一个新的细胞色素P450基因CYP6AE21。该基因含有一个1 572 bp的ORF,编码一个推定的523个氨基酸的蛋白质。CYP6AE21推定的分子量为60.5 kD,等电点为8.4,含有一个细胞色素P450的特征序列血红素结合位点区域。CYP6AE21和家蚕CYP6AE2基因一样在相同位置含有1个内含子序列,且相应的2个外显子大小完全一致。它们之间的核苷酸序列相似性达到94.5%,且在基因组上以head-to-tail的方式成簇排列,中间由约7.6 kb核苷酸序列隔开,推测它们是由其中一个基因加倍复制形成的。CYP6AE21在幼虫的头部和脂肪体,以及蛾的触角中表达量非常高;在幼虫和蛾的多个其他组织中也都有表达。P450酶系的重要组分之一细胞色素P450还原酶基因(CPR)也在雌蛾和雄蛾触角中高表达,在其它组织中都有表达,但表达量相对较低。亚细胞定位分析表明CYP6AE21表达产物定位于细胞质中,与微粒体P450经典的定位一致。推测此P450基因可能主要参与内化进细胞后的气味分子的降解清除作用。2.家蚕和野桑蚕羧酸酯酶家族基因的研究羧酸酯酶属于α/β水解酶折叠蛋白,它能有效催化含羧基酯键,酰胺键和硫酯键的内源性与外源性多种化合物的水解。关于昆虫羧酸酯酶的研究主要集中于与杀虫剂抗性的关系,而与其生理功能相关的研究比较少。本文用生物信息学方法筛选家蚕基因组数据,并通过分子手段从家蚕中鉴定了12个候选的羧酸酯酶基因序列。其中2个羧酸酯酶基因能通过选择性剪切产生多个不同的剪切变体。部分羧酸酯酶基因在同一染色体上成簇排列,其他多数基因都分散分布在不同的染色体上。根据内含子数目可以将这些基因分成两类,其中I类基因相应的内含子位点非常保守,II类基因则差异较大。同源比较和进化分析表明家蚕的羧酸酯酶之间总体差异较大,家族基因内部可能经历了较大的进化分歧。表达分析表明这些羧酸酯酶基因具有多样的组织表达特性,其中一些基因在幼虫的中肠或丝腺中高表达,一些在蛾触角中高表达。此外,2个基因BmCarE-12和BmCarE-15在幼虫脂肪体中能被脂多糖(LPS)诱导后表达升高。本研究根据这些羧酸酯酶基因相应的表达图谱和进化关系对它们可能参与的功能作用进行了探讨。家蚕BmCarE-5基因在幼虫的中肠,以及在成虫的触角等组织中都高表达。启动子分析表明该基因可能受内源激素的调控。为了进一步研究此基因的功能特性,我们利用昆虫杆状病毒表达系统分别表达了这两个基因。SDS-PAGE分析表明经BmCarE-5基因重组bacmid感染后的BmN细胞与对照组相比在预期的76 kD左右大小位置有一条特异条带。Western blot分析表明在预期的同样大小位置有一条特异条带,进一步表明BmCarE-5得到了成功表达。此基因表达产物经纯化后,进行了酶活性检测。结果表明此酶的最适温度为40℃,Km和Vmax大小分别为2.653 mM和178 OD/ min·mg protein。本研究为进一步阐明羧酸酯酶基因可能参与的生理功能提供了借鉴。为了研究酯酶与野桑蚕Bombyx mandarina和家蚕对杀虫剂抗性差异的关系,以及家蚕和野桑蚕酯酶家族基因的进化差异,采用动力学方法检测了家蚕和野桑蚕幼虫不同发育阶段,以及5龄第3 d幼虫不同组织的酯酶活性。家蚕和野桑蚕酯酶活性都在幼虫末期较高,且都具有类似的组织分布模式,在中肠中活性最高,丝腺次之,表明家蚕和野桑蚕的酯酶在进化上是相对较保守的。家蚕中肠酯酶活性是野桑蚕的1.97倍,根据“脂族酯酶突变假说”,推测野桑蚕杀虫剂抗性不是由于酯酶基因过量表达,而是由于点突变或者是由其他解毒酶系差异造成的。BmmCarE-4和BmmCarE-5共含有7个不同的选择性剪切变体,选择性剪切发生在5′端。这2个基因都存在于同一个转录单元上,且推定的氨基酸序列同源性达52%以上。其中BmmCarE-4和BmmCarE-5与家蚕相应的同源基因一样是由其中一个基因通过3′端基因序列加倍复制形成的,进一步表明家蚕和野桑蚕酯酶在分子水平上是相当保守的。丝腺酯酶活性是野桑蚕的4.49倍,其中CarE-9基因在家蚕和野桑蚕丝腺中表达量基本无差异,而CarE-7在家蚕丝腺中高表达,在野桑蚕中则几乎不表达。表明CarE-7基因在家蚕和野桑蚕丝腺中的差异表达是丝腺组织活性差异的主要原因之一,初步推测其可能在丝腺的生长发育或蚕丝合成等方面起着重要的作用。此外,野桑蚕羧酸酯酶基因BmmCarE-2与家蚕羧酸酯酶基因BmCarE-2(GenBank登录号:DQ311250)的氨基酸序列相似性最高,达98.9%。组织表达分析表明BmmCarE-2在头部和脂肪体表达量较高,在丝腺和中肠稍低,而在血液中表达量最低。3.棉铃虫羧酸酯酶基因的研究为了从分子水平上研究棉铃虫Helicoverpa armigera对杀虫剂抗性的产生机理,本文通过RT-PCR和RACE方法首次从棉铃虫中肠中克隆了一个羧酸酯酶全长cDNA序列。序列分析表明,该基因包含一个1 794 bp的开放读码框,129 bp的5′UTR和139 bp的3′UTR区域。该基因编码597个氨基酸,推测编码蛋白质的等电点PI为4.92,分子量为67.1 kD,GenBank登录号为EF547544。通过对氨基酸的同源性分析表明,该羧酸酯酶与斜纹夜蛾Spodoptera litura羧酸酯酶的同源性最高,达60﹪。半定量RT-PCR分析表明,该基因在中肠组织中表达量最高,在脂肪体和生殖腺中表达量较低,在头部则不表达。推测该羧酸酯酶基因可能主要参与棉铃虫对外源物质的解毒代谢。更多还原

【Abstract】 Many members of lepidopteran insects are agricultural and forest pests, which cause immeasurable losses every year all over the world. Wide use of insecticides has resulted in increased insecticide resistance in many pests, and has then caused great difficulties to control these pests.Cytochrome P450 is a superfamily of heme proteins, which partipicates in oxidative metabolism of endogenous and exogenous compounds. In insects, a substantial amount of research has indicated that cytochrome P450 enzyme system plays pretty important roles in detoxifying metabolism of pyrethroid insecticides and plant toxins. Carboxylesterase belongs toα/βhydrolase fold protein family, which comprise of a multigene family and can effectively hydrolyze the endogenous and exogenous compounds containing carboxylester, amide, and thioester bonds. In insects, the research about carboxylesterase is mainly focused on its involvement in insecticide resistance. Moreover, many studies have demonstrated that many metabolic enzymes such as cytothrome P450 and carboxylesterase are involved in the degradation of odorants.Bombyx mori is another insect whose whole genome sequences have been fully completed after Drosophila melanogaster and Anopheles gambiae. It has been considered as the lepidopteran model insect and is also the only whole genome sequence available lepidopteran insect. A great number of genome sequences and EST data have provided great convenience to study new genes and their corresponding functions. Bombyx mori and Bombyx mandarina originated from the same ancestor, and belong to family Bombycidae, order Lepidoptera. There have been great differences in insecticide resistance between the two species due to different selective pressures of insecticides. To research detoxifying metabolism and olfactory detection related cytochrome P450 and carboxylesterase family genes will provide clues to the control of pests such as lepidopteran insects, and facilitate the research about the physiological functions of the two family genes and evolutionary differences of species under domestication. In this research, multiple cytochrome P450 and carboxylesterase genes were identified by using bioinformatic and molecular approaches. The evolutionary relationships between these genes were analyzed at the molecular level. Tissue expression and developmental expression profiles were studied, as well as the inductive effects of xenobiotics on these genes. Some genes were expressed by using insect baculovirus express system, and the enzymatic activity of a carboxylesterase gene product was further assayed. We first compared the differences of esterases between Bombyx mori and Bombyx mandarina at the biochemical and molecular levels. A first carboxylesterase gene was also cloned from the agricultural pest, Helicoverpa armigera.1. The research of cytochrome P450 genes from Bombyx moriIn this study, we cloned two CYP4 and four CYP6 genes using bioinformatics and RT-PCR approaches. Sequence analysis showed that these genes contained conserved P450 gene sequence regions and one conserved intron. CYP4M5 and CYP4M9 were clustered together in a head-to-tail arrangement possibly due to gene duplication. Blast analysis showed that these P450 genes shared significant similarity with CYP4 and CYP6 families involved in the metabolism and detoxification of xenobiotics in other insects. RT-PCR analysis showed that these P450 genes were all highly expressed in the midgut or fat body of silkworms. Some of the P450 gene expression was increased while expression of other P450 genes was decreased as the instar age increased. When the larvae were exposed to 1.75×10-5 % of cypermethrin, 3.5×10-6 % of cypermethrin and 0.1 % of rutin, expression of CYP6AB5 was increased by 2.3-fold, 2.2-fold and 1.9-fold, respectively, while exposure to 0.1 % quercetin did not influence the expression of CYP6AB5. In contrast, expression of the other five P450 genes was suppressed after exposure to these compounds.CYP6AB5 gene was expressed by using insect Bac-to-Bac baculovirus expression system. The expression of this gene lay the foundation for characterization of substrate specificity of this enzyme by further constructing in vitro metabolizing enzymatic system.Signal inactivation is a critical step in the olfactory dynamic process, which involves various odorant-degrading enzymes. In this study, a cytochrome P450 cDNA CYP6AE21 was cloned from the moth antenna of Bombyx mori by RT-PCR method. CYP6AE21 contains a 1 572 bp ORF, which encodes a putative protein of 523 amino acids. This putative protein have a calculated molecular mass of 60.5 kD, pI of 8.4, and a P450 characteristic structure heme-binding region. CYP6AE21 and Bombyx mori CYP6AE2 both contain only one intron which exists at the same site, and their relative two exons have exactly the same size. The two genes share 94.5% nucleotide similarity, and cluster together on the same chromosome in a head-to-tail arrangement separated by a approx. 7.6 kb intergenic region. CYP6AE21 was highly expressed in larval head and fat body, and moth antenna. It was also detected in many other larval and moth tissues. NADPH P450 reductase (CPR), a very important component of monooxygenase system, was also highly expressed in the moth antenna, and detected in other moth tissues with low levels. Subcellular localization analysis showed that the expression product of CYP6AE21 is localized in cytoplasm, which is consistent with the classical localization of microsomal P450s. It was postulated that this P450 gene might participate in the degradation of odorants after their internalization into cells.2. The research of carboxylesterase genes from Bombyx mori and Bombyx mandarinaCarboxylesterase belongs toα/βhydrolase fold protein family, which can effectively catalyze the hydrolysis of endogenous and exogenous compounds containing carboxylester, amide and thioester bonds. In insects, the research of carboxylesterase is mainly focused on its involvement in insecticide resistance. Whereas, there is few study related to its physiological functions. Bioinformatics method was used to screen the genome sequence of Bombyx mori, and 12 candidate carboxylesterase genes were identified through molecular approaches. Two of the 12 candidate genes can generate various variants through alternative splicing. Some of these identified genes are clustered together on the chromosomes, while others are distributed among different chromosomes. These carboxylesterase genes can be divided into two groups according to the number of their introns. The 1 group contains conservative corresponding intron sites; the 2 group contains introns with great varieties. Homology comparison and phylogenetic analysis showed that there are great differences among these sequences, suggesting that they must have undergone great evolutionary divergences. Expression analysis indicated that some of these carboxylesterase genes were dominantly expressed in midgut or silk gland, while some others were detected at high levels in moth antenna. Moreover, BmCarE-12 and BmCarE-15 transcripts were increased in fat body after the induction by lipopolysaccharide (LPS). The putative functions of these carboxylesterase genes were discussed with regard to their respective expression patterns and phylogenetic relationships.BmCarE-5 was expressed at high levels in larval midgut, and moth antenna. Promoter analysis showed that the expression of this gene might be regulated by endogenous hormones. In order to research its potential function, we expressed this gene by using insect baculovirus expression system. SDS-PAGE analysis showed that there is a specific band of about 76 kD at the lane of BmCarE-5 recombinant bacmid infected BmN cells compared with control group. Western blot analysis showed that there is also a specific band at the same position, indicating that BmCarE-5 was successfully expressed. Enzymatic activity of this enzyme was assayed after purification of its expression product. The results showed that the optimum temperature for BmCarE-5 is 40℃, and Km and Vmax for this enzyme are 2.653 mM and 178 OD/min·mg protein, respectively. This research will facilitate the characterization of the physiological function of carboxylesterase genes.In order to research the realtionship between esterases and the insecticide resistance differences between Bombyx mandarina M. and Bombyx mori L., and the evolutionary difference between esterases from the two species, esterase activities at different larval stage and different tissues of day 3 fifth-instar larvae of Bombyx mori and Bombyx mandarina were detected using the kinetic method. The esterase activities of Bombyx mori and Bombyx mandarina were both higher at late larval stage and had similar tissue distributions with highest values in midguts and less in silk glands, suggesting that the esterases of Bombyx mori and Bombyx mandarina were relatively conserved during the course of evolution. The esterase activity in the midgut of Bombyx mori was 1.9-fold higher than that in Bombyx mandarina. According to the mutant ali-esterase hypothesis, the insecticide resistance of Bombyx mandarina was possibly not caused by overexpression of esterases, but probably through point mutations or other detoxifying enzymes. BmmCarE-4 and BmmCarE-5 had 7 variants caused by splicing differently at 5′terminal. The two genes were located on the same transcription unit, and shared over 52% amino acid identity. BmmCarE-4 and BmmCarE-5 originated from the duplication of 3′terminal sequence of one of the two genes in a pattern similar to the two orthologous genes in Bombyx mori, which further demonstrates the high conservation of esterases at the molecular level between the two species. The esterase activity in the silk gland of Bombyx mori was 4.49-fold higher than that in Bombyx mandarina. The expression level of CarE-9 in silk gland was nearly identical between Bombyx mori and Bombyx mandarina. Whereas, CarE-7 was expressed highly in silk gland of Bombyx mori, but nearly not detected in Bombyx mandarina. The expression variance of CarE-7 in silk glands of Bombyx mori and Bombyx mandarina was one of the main reasons for the difference in enzymatic activities in these tissues, and this gene might play a role in the growth and development of silk gland or the synthesis of silk protein. Moreover, BmmCarE-2 shared highest amino acid similarity (98.9%) with BmCarE-2 from Bombyx mori (Genbank accession number: DQ311250). Tissue expression analysis demonstrated that BmmCarE-2 was expressed highly in head and fat body, slightly lower in silk gland and midgut, and extremely low in hemolymph.3. The research of carboxylesterase gene from Helicoverpa armigeraIn order to study the molecular mechanisms of insecticide resistance in Helicoverpa armigera, a first full-length carboxylesterase cDNA was cloned from Helicoverpa armigera through RT-PCR and rapid amplification of cDNA ends (RACE) strategies. Sequence analysis revealed that this gene contains a 1 794 bp ORF, a 129 bp 5′UTR and 139 bp 3′UTR, which encodes a 597 amino acid protein. The predicted molecular weight and isoelectric point of this carboxylesterase were 67.1 kD and 4.92, respectively. This gene was deposited in GenBank under the accession number EF547544. Homology analysis showed that this carboxylesterase is most similar to the carboxylesterase from Spodoptera litura with 60﹪amino acid identity. Semi-quantitative RT-PCR showed that this gene was highly expressed in midgut, low-expressed in fat body and gonad, and not expressed in head. It was postulated that this carboxylesterase gene may be involved in the detoxification of xenobiotics.更多还原