草地贪夜蛾解毒代谢相关基因家族的进化分析

近年来草地贪夜蛾Spodoptera frugiperda在世界各地猖獗为害,其寄主范围广,且已对多种化学药剂和转Bt玉米产生抗性。本文对包括草地贪夜蛾在内的14种昆虫进行了解毒代谢相关基因家族的注释,在草地贪夜蛾中发现了152个细胞色素P450(cytochrome p450,P450)、92个ABC转运蛋白(ATP-binding cassette transporter,ABC transporter)、44个谷胱甘肽转移酶(glutathione s-transferase,GST)以及39个尿苷二磷酸葡萄糖醛酸转移酶(UDP-glucuronosyl-transferase,UGT)。与其他鳞翅目物种相比,草地贪夜蛾的P450和GST基因数量最多,其中152个P450基因可被归类至Mito、CYP2、CYP3和CYP4四个簇中;Mito和CYP2簇的P450基因总数与家蚕Bombyx mori相近,但CYP3和CYP4簇的P450基因明显多于家蚕,进化分析结果显示CYP3和CYP4簇的P450基因发生了明显的扩增;与家蚕P450基因的共线性分析发现草地贪夜蛾89个P450基因在其基因组上呈簇分布并发生了簇的扩增;与家蚕相比,GST和ABC转运蛋白家族基因的数量明显增多。UGT基因家族没有明显增加,但在基因进化分析中有成簇现象,表明可能存在近期的扩增事件。本研究首次从基因组水平鉴定了草地贪夜蛾解毒代谢相关基因家族,对理解草地贪夜蛾的生物学特性及抗性的形成具有重要指导意义。     

家蚕细胞色素P450的基因组学分析

细胞色素P450参与许多基础代谢过程, 确保有机体避免外源复合物对它们的伤害. 将预测的家蚕基因同已知的P450基因进行蛋白质序列比对, 在家蚕基因组中发现86个可能的细胞色素P450基因, 初步将它们归于32个P450亚家族. 通过比较基因组学分析, 发现细胞色素P450基因在果蝇与家蚕中的分布规律具有总体上的相似性, 但在某些P450基因家族中的分布也有差异. 特别是在CYP4A, CYP4C, CYP4D, CYP6A, CYP6AE, CYP6B和CYP9A等7个亚家族中P450s差异分布更明显. 进一步将这些P450基因的DNA序列与家蚕ESTs进行核酸序列比对, 其中49个可能P450基因发现有ESTs证据, 证明了这些基因在转录水平的真实性.     

家蚕P450基因CYP18A1的克隆、序列分析及转录活性

蜕皮激素对昆虫生长、发育和繁殖有重要调控作用,尤其对蜕皮和变态过程。利用GenBank上登录的蜕皮激素C₂₆羟基化酶候选基因CYP18A1的氨基酸序列对家蚕Bombyx mori全基因组数据库进行BLASTP比对,发现了家蚕直向同源基因(ortholog),其完全编码序列经RT-PCR检测和克隆、测序验证后,再以此为信息探针检索家蚕表达序列标签(expressed sequence tags,EST)数据库进行拼接延伸,获得了一条包括5′非翻译区在内的长度为1 737 bp的cDNA序列,验证结果也表明与电子克隆序列完全一致(GenBank登录号为EF421988,P450命名委员会将其命名为CYP18A1)。该基因的开放阅读框为1 623 bp,编码541个氨基酸,含有包括P450s特征结构域在内的所有昆虫P450基因的5个保守结构域,其推定的分子量为61.67 kD,等电点为 8.54。将该基因cDNA序列与家蚕基因组序列进行比对,结果表明该基因具有6个外显子,5个内含子,外显子／内含子边界符合经典的GT-AG规则。同源性分析也发现家蚕CYP18A1与其他昆虫的直向同源基因具有较高相似性。用RT-PCR方法对家蚕主要发育变态时期与组织进行检测,显示出该基因的转录表达不仅具有时空特异性,而且在表达时期上与已报道的蚕体内蜕皮激素含量变化有紧密的一致性。该研究进一步证实了CYP18A1基因与昆虫体内蜕皮激素代谢平衡相关联。     

家蚕Hox基因研究进展

Hox基因（homeobox genes）在昆虫躯体模式（body plan）的发育调控机制中扮演着重要角色,其表达具有严格的组织特异性和胚胎发育的程序性。家蚕Bombyx mori作为鳞翅目昆虫的代表,其Hox基因也陆续得到鉴定。在家蚕中存在一个拟复等位基因群--E群基因,其突变表型均与过剩斑纹和过剩附肢有关,这可能与Hox基因有着密切联系。家蚕全基因组测序完成后,发现其Hox基因簇中存在12个特有的homeobox基因（Bmshx1~Bmshx12）, 说明家蚕Hox基因可能具有独特的生物学意义。我们还利用家蚕基因芯片数据分析了Bmlab与Bmpb基因的组织表达特征。通过对家蚕Hox基因的研究,探索家蚕躯体模式建立机制,可望为解析其他鳞翅目昆虫的躯体模式的建立机制提供理论依据。本文就家蚕Hox基因的表达、功能及其与E群突变的关系等方面进行了综述。     

昆虫细胞色素P450基因的多样性、进化及表达调控

细胞色素P450单加氧酶（cytochrome P450 monooxygenases, P450s）是由多个功能相关的亚铁血红素 硫醇盐蛋白基因组成的一个基因超家族, 在各种内源和外源物质的代谢中起着主要作用。目前GenBank中注册的昆虫P450基因序列已超过1 000个, 其中双翅目占序列总数的74%, 鳞翅目占序列总数的16%。而昆虫P450基因序列已克隆的全长序列中大部分属于CYP4和CYP6家族, 两个家族成员分别占总数的20%和45%。利用GenBank中现已注册的昆虫P450基因的cDNA全长序列进行比对并绘制进化树, 揭示不同种类昆虫P450的亲缘关系。结果显示基于P450基因的昆虫部分目的进化关系与大部分先前依据其他分子数据或形态分类学得到的昆虫系统进化关系基本吻合。现有研究表明, 细胞色素P450基因的表达可能受顺式作用元件(cis-acting element)、反式作用因子(trans-acting factor)或两者共同调控, 调控可能涉及转录增强的转录机制或mRNA稳定性增加的转录后机制。     

CPR和P450基因在昆虫抗药性中的作用研究进展

P450酶系在昆虫代谢农药中有重要作用,NADPH-细胞色素P450还原酶（NADPH-cytochrome P450 reductase,CPR）和细胞色素P450（P450）在该酶系起核心作用。昆虫具有P450超基因家族,但只有一个单一的CPR基因,CPR是昆虫所有参与农药代谢的P450酶的唯一电子供体,其影响P450活性。P450基因的高水平表达在害虫抗药性中具有重要作用,P450基因介导的昆虫抗药性是最重要的代谢抗性类型。不同P450基因的高表达的调控机制不同,引起P450基因过量表达的原因可能有P450基因的编码区突变、顺式作用元件和反式作用因子变化、基因扩增等。细胞色素P450介导的抗药性存在一定程度的进化可塑性,即同种昆虫不同种群对相同的农药产生抗药性时,导致抗性产生的P450基因不同;同一昆虫品系在某种农药的抗性选择压力下,影响抗性的P450基因的种类和表达特性会随着持续的农药选择而发生变化。最近的研究显示,CPR的变异和昆虫抗药性相关,但是昆虫CPR基因介导抗药性的机制还缺乏深入研究。全面阐释P450酶系介导昆虫抗药性的机制、建立基于P450基因表达量变化与CPR突变的抗性分子标记,对于害虫抗药性治理具有重要意义。     

灰飞虱唾液腺三大解毒酶家族的转录组分析

灰飞虱Laodelphax striatellus （Fallén）是危害多种禾本科经济作物的重要刺吸式害虫。唾液腺对刺吸式口器昆虫取食植物尤其重要, 其分泌的唾液可以帮助刺吸式口器昆虫刺穿植物、 消化食物、 解毒植物的次生物质。细胞色素P450单加氧酶（cytochrome P450 monooxygenase, P450）、 谷胱甘肽S 转移酶（glutathione S transferase, GST）和羧酸酯酶（carboxylesterase, CarE）是昆虫主要的解毒酶系。为了分析解毒酶基因在灰飞虱唾液腺中的表达谱, 本研究对灰飞虱成虫唾液腺进行转录组测序、 重头组装和注释, 并与豌豆蚜Acyrthosiphon pisum和西方蜜蜂Apis mellifera的同源蛋白进行系统发育分析。发现有9个谷胱甘肽S 转移酶（glutathione S transferase, GST）基因、 22个羧酸酯酶（carboxylesterase, CarE）基因和39个细胞色素P450单加氧酶（cytochrome P450 monooxygenase, P450）基因在灰飞虱唾液腺中表达。通过对同源蛋白进行系统发育分析, 发现灰飞虱唾液腺大部分的CarE是参与消化/解毒和激素/信息素的加工, 而参与神经/发育的CarE很少; 灰飞虱唾液腺表达的P450基因远远少于豌豆蚜和西方蜜蜂基因组的P450基因数, 且只有CYP6和CYP4家族的成员; GST家族在3种昆虫的保守性最高。研究结果为灰飞虱对寄主植物和杀虫剂的适应性研究奠定了基础。     

小菜蛾CYP6基因序列分析及其进化地位探讨

利用简并引物和特异性引物,通过RT-PCR和RACE技术,克隆出了一个小菜蛾细胞色素P450CYP6基因。该基因全长1661bp,编码区在26~1570位,翻译514个氨基酸,GenBank登录号为AY971374。分析该基因在昆虫CYP6基因家族系谱树中的位置和5′-UTR的序列,可以认为,小菜蛾CYP6基因与同为鳞翅目的其他昆虫进化地位相当,而明显与相对低等的蜚蠊目昆虫的同家族基因亲缘关系远。     

西花蓟马细胞色素P450基因cDNA片段克隆与mRNA表达分析

细胞色素P450介导的解毒作用增强是昆虫对杀虫剂产生抗性的重要机制。本文通过RT-PCR克隆了西花蓟马CYP4家族5个细胞色素P450基因c DNA片段。多重序列比对发现,这5个基因在氨基酸水平的一致性在40%-76%之间。采用实时荧光定量PCR对5个CYP4基因的mRNA表达水平的分析发现,阿维菌素抗性品系CYP4-1、CYP4-2和CYP4-5的表达水平分别是敏感品系的3.50、4.00和2.48倍,表明西花蓟马对阿维菌素的抗性可能与这3个CYP4基因的过量表达相关。     

五种鳞翅目害虫中抗药性相关基因的转录组学分析

随着测序技术的发展,昆虫转录组数据不断积累,在昆虫学研究中的应用也越来越广泛。在害虫抗药性的研究中,转录组数据分析也是重要的最新研究手段。本文通过对5种鳞翅目害虫转录组的生物信息学分析,鉴定出13种与抗药性相关的基因,发现细胞色素P450基因序列数量最多,共887条。另外,谷胱甘肽S-转移酶、鱼尼丁受体、氨肽酶N、糖基转移酶相关的基因序列多于100条。同时,对这5种鳞翅目昆虫中部分Bt受体相关的基因做了多序列比对和进化分析。分析认为,从多物种、多基因的角度提出对农药抗性的系统性研究,是害虫抗性研究的发展趋势。     

Genome-wide analysis of cytochrome P450 monooxygenase genes in the silkworm, Bombyx mori

Based on the advances in the silkworm genome project, a new genome-wide analysis of cytochrome P450 genes was performed. A total of 84 CYP-related sequences were identified and could be classified into 26 families and 47 subfamilies according to standard nomenclature. Seventy eight of the eighty four genes appear to be functional and six are probable pseudogenes. The distribution of Bombyx mori P450s in the genome shows that most of them are tandem arranged on chromosomes, only 34 genes are present as singletons, with 8 clusters including 3 or more than 3 genes. Sequence alignments were used to reconstruct phylogenetic trees and to analyze the intron-exon organizations of the functional genes. The conserved intron positioning agrees perfectly with their common grouping on the tree. The presence of three extremely ancient introns which are conserved across different clans indicates that a few introns are still highly conserved after they have undergone extensive evolutionary changes of B. mori P450 duplication and divergence. Comparison of the P450s from B. mori to the P450s from Drosophila melanogaster shows that the expansion is not uniform across the gene families. Remarkably, two mitochondrial families, the B. mori CYP333 and D. melanogaster Cyp12, formed two orthologous groups in the phylogenetic tree. All CYP333s can be proposed to be related to xenobiotic metabolism in accordance with the D. melanogaster Cyp12s. The characterization and evolutionary analysis of P450s from B. mori in the current study provide useful information for understanding the characteristics and diversity of P450s from B. mori and the baseline for functional analyses of individual P450s in this model Lepidopteran insect.     

Analysis of cytochrome P450 genes in silkworm genome (Bombyx mori)

Cytochrome P450 monooxygenases (P450) are im-portant metabolic enzymes involved in the metabolism not only of a wide range of endogenous compounds such as fatty acids, steroids, hormones or vitamins, but also of exogenous substrates such as drugs, chemicals including environmental pollutants, such carcinogens as polycyclic aromatic hydrocarbons, and pesticides[1]. P450s are found virtually in all aerobic organisms, including organisms as diverse as in insects, plants, mammals, birds and bacter…     

Cytochrome P450 and actin genes expressed in Helicoverpa zea and Helicoverpa armigera: paralogy/orthology identification, gene conversion and evolution.

Molecular phylogenetic analysis was conducted using conserved cytoplasmic actin and diversified cytochrome P450 (P450) sequences isolated from Helicoverpa zea and Helicoverpa armigera, two species thought to be closely related based on allozyme analyses. These sequences were compared in turn with published sequences from other insects to gain insight into how different gene families evolve. In Bombyx mori and these Helicoverpa species, cytoplasmic actin genes are present as a pair of tandemly duplicated paralogs with coding sequence identities as high as 95.5% (B. mori), 98.9% (H. zea) and 98.5% (H. armigera) due to recent 5'-polar gene conversions. Phylogeny and interspecies comparisons assign the six actin genes into two orthologous groups: HaA3a/HzA3a/BmA3 and HaA3b/HzA3b/BmA4, which exhibit more similarities between H. zea and H. armigera than between Helicoverpa species and B. mori. Like the actin genes in H. zea, four CYP6B genes exist as two pairs of duplicated paralogs with recent 5'-polar gene conversions. Interspecific comparisons and phylogeny analysis identified three groups of orthologous CYP6B genes: H. zea CYP6B8 or CYP6B28/H. armigera CYP6B7, H. zea CYP6B27/H. armigera CYP6B6, and H. zea CYP6B9/H. armigera CYP6B2/Heliothis virescens CYP6B10. The low degree of divergence in the first two of these groups is comparable to allelic variation within a single species. These orthologous relationships and the high degrees of similarity in both actin and P450 genes strongly indicate that these Helicoverpa species are extremely closely related.     

家蚕细胞色素P450基因CYP6AE21的克隆、表达分析及亚细胞定位

信号失活是嗅觉动态过程的一个重要步骤, 这一过程涉及多样的气味降解酶类。本研究利用RT-PCR方法从家蚕Bombyx mori雄蛾的触角中克隆了一个细胞色素P450基因CYP6AE21。该基因含有一个1 572 bp的开放阅读框（open reading frame, ORF）, 编码523个氨基酸, 预测分子量为60.5 kD, 等电点为8.4, 含有细胞色素P450的特征序列血红素结合位点区域。CYP6AE21和CYP6AE2基因一样在相同位置含有1个内含子序列, 且相应的2个外显子大小相同。两者的核苷酸序列相似性达到94.5%, 且在基因组上以头尾相连的方式成簇排列, 中间由约7.6 kb核苷酸序列隔开。CYP6AE21在幼虫的头部和脂肪体, 以及雄蛾和雌蛾的触角中表达量较高; 在幼虫的其他组织和蛾的多个组织中也有一定的表达。P450酶系的重要组分之一--NADPH细胞色素P450还原酶（cytochrome P450 reductase, CPR）基因也在雌蛾和雄蛾触角中高水平表达, 而在其他组织中表达量相对较低。亚细胞定位分析表明CYP6AE21表达产物定位于细胞质中。推测CYP6AE21和CYP6AE2是由其中一个基因加倍复制形成的; 此P450的功能之一可能是参与内化进细胞的气味分子的降解清除。     

玉米螟P450基因cDNA的克隆及植物次生物质对其诱导表达

昆虫细胞色素P450是一类在生物代谢中起重要作用的基因家族,承担着许多重要的生 理功能,包括对植物次生物质代谢和杀虫剂解毒等.本研究以玉米螟5龄幼虫中肠的总RNA为 模板,根据不同昆虫P450基因家族保守氨基酸序列,设计并合成简并引物,利用RT-PCR扩 增出了玉米螟细胞色素P450基因cDNA片段,将其克隆到pMD19 T载体进行序列测定.测序获得了编码213个氨基酸残基的641 bp的DNA片段,命名为OfP450(GenBank登录号：EU807990) ;与已公布的棉铃虫、家蚕、欧洲防风草结网毛虫、小菜蛾和黑腹果蝇等的细胞色素P450氨基酸序列进行比较,其一致性分别为55％、50％、49％、44％和31％.将植物次生物质棉酚、丁布添加入人工饲料中,对玉米螟进行了饲喂实验,采用优化半定量RT-PCR,以18S rRNA为内标,RT-PCR检测进食棉酚、丁布植物化合物的玉米螟中肠P450基因的转录. 结果表明,其转录水平能被棉酚、丁布显著诱导. 提示本实验克隆的玉米螟细胞色素P450对植物次生物质的代谢作用与P450表达量呈正相关.该研究为下一步将植物介导的RNA干扰技术应用于害虫生物防治提供坚实的基础.     

Synthesis and phosphorylation of ecdysteroids during ovarian development in the silkworm, Bombyx mori

In the silkworm, Bombyx mori, it has been demonstrated that most free ecdysteroids in the ovary are converted to physiologically inactive ecdysteroid 22-phosphates, which are then transformed back to free ecdysteroids during early embryonic development. Two specific enzymes involved in the reciprocal conversion of ecdysteroids, namely, ecdysteroid 22-kinase (EcKinase) and ecdysteroid-phosphate phosphatase, have been isolated and characterized. In this study, we first attempted a phylogenetic analysis of EcKinase. The resulting phylogenetic tree showed that many proteins homologous to B. mori EcKinase are found not only in ecdysozoa, including insects and nematodes, but also in teleosts, fungi, and bacteria. We then investigated the sites where free ecdysteroids are synthesized and phosphorylated in the ovary. We found that (1) the mRNAs of two P450 enzymes involved in ecdysteroidogenesis, CYP306a1 (25-hydroxylase) and CYP314a1 (20-hydroxylase), are expressed mainly in follicle cells, (2) EcKinase mRNA localizes in the oocyte and nurse cells, and (3) EcKinase immunoreactivity localizes mainly in the external region of the oocyte, not in nurse cells or follicle cells. From these results, we suggest that ecdysteroids in the B. mori ovary are synthesized in follicle cells and transferred into the oocyte, where they are phosphorylated by EcKinase, whose mRNA originates from nurse cells and the oocyte itself.     

Cytochromes P450: Roles in Diseases

The cytochrome P450 superfamily consists of a large number of heme-containing monooxygenases. Many human P450s metabolize drugs used to treat human diseases. Others are necessary for synthesis of endogenous compounds essential for human physiology. In some instances, alterations in specific P450s affect the biological processes that they mediate and lead to a disease. In this minireview, we describe medically significant human P450s (from families 2, 4, 7, 11, 17, 19, 21, 24, 27, 46, and 51) and the diseases associated with these P450s.