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。     

昆虫细胞色素P450基因的克隆及其策略

本记述了目前已克隆的105个昆虫细胞色素P450基因cDNA和片段,它们分属CYP4、CYP6、CYP9、CYP12、CYP18和CYP28等6个家族：同时,综述和分析了克隆这些基因、cDNA和片段所采用的策略及其优缺点。     

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.     

桔小实蝇CYP4D46的克隆及其组织特异性表达研究

从桔小实蝇Bactrocera dorsalis(Hendel)成虫体内提取总RNA,利用RT-PCR和cDNA末端快速扩增技术获得了一个新的细胞色素P450基因cDNA序列全长.该基因经细胞色素P450基因命名委员会命名为CYP4D46(Gen-Bank登录号:GU292422),其cDNA全长为1717 bp,包含1530 bp的完整开放阅读框(ORF),编码510个氨基酸,理论分子量约为58.40 kD,等电点为8.82.系统发育分析表明该基因与昆虫第4家族P450基因具有较高的同源性.实时定量PCR分析发现,CYP4D46基因在脂肪体中的相对表达量较高,分别是马氏管和中肠内的756倍和60倍,说明CYP4D46可能与脂肪体的重要生理功能相关.     

甘蓝型油菜BnCYP79B1基因的克隆与表达

硫苷是十字花科植物的一种次生代谢产物,其合成途径受细胞色素P450的CYP79家族蛋白的调控,该实验采用同源克隆技术在甘蓝型油菜中克隆到了CYP79B1基因,命名为BnCYP79B1(GenBank登录号为JX535391.1)。BnCYP79B1基因cDNA全长1 625bp,编码一个含有541个氨基酸、理论等电点为8.88。序列对比结果显示,BnCYP79B1与花椰菜CYP79B1在DNA序列上的相似性为98.83%,推测蛋白氨基酸序列的相似性为99.26%。通过不同时期不同部位BnCYP79B1基因表达量的分析,发现BnCYP79B1基因在高秆高硫苷品系的根中表达量较高,而对矮秆高硫苷品系则是叶中表达量较高。在BnCYP79B1表达总量上,高秆品系较矮秆品系高,高硫苷品系较低硫苷品系高。     

孟氏隐唇瓢虫细胞色素P450基因的克隆与序列分析

采用同源克隆和RACE技术,从孟氏隐唇瓢虫Cryptolaemus montrouzieri Mulsant中克隆到细胞色素P450CYP9Z401基因的c DNA全序列(Gen Bank number:KP164813)。c DNA全长1722 bp,包含3'非编码区域(UTR)为86 bp和5'UTR为49 bp,开放阅读框(ORF)长1587 bp,编码528个氨基酸。预测的分子量为61.27 k D,理论等电点为6.58,无信号肽结构,在2-20氨基酸处存在跨膜螺旋。该基因拥有细胞色素P450特征序列螺旋C、螺旋K和Meander区域,另外还有血红素结合区(1个氨基酸差异)和螺旋I区(2个氨基酸差异)。与其他昆虫的CYP9家族基因比较,与赤拟谷盗Tribolium castaneum同源性最高,为47%,系统发育分析也表明两者的亲缘关系最近。CYP9Z401基因的克隆和比较分析为进一步深入研究孟氏隐唇瓢虫的细胞色素P450基因功能及其进化具有重要意义。     

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。     

苹果蠹蛾细胞色素P450基因CYP332A19和CYP337B19的克隆及表达分析

【目的】本研究旨在通过克隆苹果蠹蛾Cydia pomonella细胞色素P450基因CYP332A19和CYP337B19,并对其进行序列和表达分析,以更好地了解这两个P450基因在植物次生物质解毒方面的作用,为进一步的功能研究提供依据。【方法】采用本地BLAST搜索苹果蠹蛾转录组数据库获得细胞色素P450基因cDNA序列,采用RT-PCR技术克隆目的基因的编码区。利用生物信息学软件分析目的基因的序列特征及与其他近缘物种的P450基因的系统进化关系。采用RT-qPCR技术测定目的基因在苹果蠹蛾不同发育阶段(卵、1-5龄幼虫、蛹和成虫)、4龄幼虫不同组织(头部、表皮、脂肪体、中肠和马氏管)以及4龄幼虫分别取食添加0.1%香豆素和0.5%槲皮素的人工饲料2 d后的表达水平。【结果】克隆获得苹果蠹蛾细胞色素P450基因CYP332A19(GenBank登录号:MF574708)和CYP337B19(GenBank登录号:MF574697)的全长cDNA序列,开放阅读框(ORF)分别长1 518和1 491 bp,分别编码505和496个氨基酸,其蛋白质分子量分别为58.586和57.734 ...     

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。     

小菜蛾细胞色素P450基因的克隆及生物信息学分析

利用RT—PCR技术,从小菜蛾体内克隆了细胞色素P450基因CYP6序列,GenBank登录号为AY971374。生物信息学技术分析表明,该序列由1661bp组成,熔点102℃,退火温度87℃,单链分子质量539．83kDa,双链分子质量1079．65kDa,可以翻译514个氨基酸,组成的蛋白质分子式C2682H4154N702O747S30,分子量是59146．5,总原子数8315,等电点pl为8．63,高级结构与Cytochrome P450 Bm-3基因有较高的相似性。     

Permethrin Induction of Multiple Cytochrome P450 Genes in Insecticide Resistant Mosquitoes,Culex quinquefasciatus

The expression of some insect P450 genes can be induced by both exogenous and endogenous compounds and there is evidence to suggest that multiple constitutively overexpressed P450 genes are co-responsible for the development of resistance to permethrin in resistant mosquitoes. This study characterized the permethrin induction profiles of P450 genes known to be constitutively overexpressed in resistant mosquitoes, Culex quinquefasciatus. The gene expression in 7 of the 19 P450 genes CYP325K3v1, CYP4D42v2, CYP9J45, (CYP) CPIJ000926, CYP325G4, CYP4C38, CYP4H40 in the HAmCqG8 strain, increased more than 2-fold after exposure to permethrin at an LC50 concentration (10 ppm) compared to their acetone treated counterpart; no significant differences in the expression of these P450 genes in susceptible S-Lab mosquitoes were observed after permethrin treatment. Eleven of the fourteen P450 genes overexpressed in the MAmCqG6 strain, CYP9M10, CYP6Z12, CYP9J33, CYP9J43, CYP9J34, CYP306A1, CYP6Z15, CYP9J45, CYPPAL1, CYP4C52v1, CYP9J39, were also induced more than doubled after exposure to an LC50 (0.7 ppm) dose of permethrin. No significant induction in P450 gene expression was observed in the susceptible S-Lab mosquitoes after permethrin treatment except for CYP6Z15 and CYP9J39, suggesting that permethrin induction of these two P450 genes are common to both susceptible and resistant mosquitoes while the induction of the others are specific to insecticide resistant mosquitoes. These results demonstrate that multiple P450 genes are co-up-regulated in insecticide resistant mosquitoes through both constitutive overexpression and induction mechanisms, providing additional support for their involvement in the detoxification of insecticides and the development of insecticide resistance.     

CYP52 (cytochrome P450alk) multigene family in Candida maltosa: molecular cloning and nucleotide sequence of the two tandemly arranged genes

Southern blot analysis under low-stringency conditions using a previously isolated n-alkane-inducible cytochrome P450 (P450alk) gene as a probe revealed the presence of multiple P450alk-related genes in the genome of Candida maltosa. Nine P450alk-related genes (one reported previously and eight in the present report) were isolated from a genomic library constructed from this strain, and these were classified on the basis of sequence similarities into three pairs of putative allelic genes and three nonallelic genes. Two pairs of these alleles were tandemly arranged in the genome. The complete nucleotide sequences of one of these pairs were determined and compared to other members of this P450 family (CYP52) in C. maltosa and C. tropicalis. Northern blot analysis further showed that these genes were regulated by carbon sources. These results provide evidence for a P450alk (CYP52) multigene family in C. maltosa.     

The cytochrome P450 gene superfamily in Drosophila melanogaster: annotation, intron-exon organization and phylogeny

The cytochrome P450 gene superfamily is represented by 90 sequences in the Drosophila melanogaster genome. Of these 90 P450 sequences, 83 code for apparently functional genes whereas seven are apparent pseudogenes. More than half of the genes belong to only two families, CYP4 and CYP6. The CYP6 family is insect specific whereas the CYP4 family includes sequences from vertebrates. There are eight genes coding for mitochondrial P450s as deduced from their homology to CYP12A1 from the house fly. The genetic map of the distribution of D. melanogaster P450 genes shows (a) the absence of P450 genes on the chromosome 4 and Y, (b) more than half of the P450 genes are found on chromosome 2, and (c) the largest cluster contains nine genes. Sequence alignments were used to draw phylogenetic trees and to analyze the intron-exon organization of each functional P450 gene. Only five P450 genes are intronless. We found 57 unique intron positions, of which 23 were phase zero, 19 were phase one and 15 were phase two. There was a relatively good correlation between intron conservation and phylogenetic relationship between members of the P450 subfamilies. Although the function of many P450 proteins from vertebrates, fungi, plants and bacteria is known, only a single P450 from D. melanogaster, CYP6A2, has been functionally characterized. Gene organization appears to be a useful tool in the study of the regulation, the physiological role and the function of these P450s.     

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.     

Spatially distinct expression of two new cytochrome P450s in leaves of Nepeta racemosa/: identification of a trichome-specific isoform

Using a PCR-based approach, two novel cytochrome P450 cDNAs were isolated from a catmint (Nepeta racemosa) leaf cDNA library. The cDNAs (pBSK3C7 and pBSK4C3) were 76.9% identical in their nucleotide sequences, indicating that they are the products of two closely-related genes. A comparison of the sequence of these cDNAs with database sequences indicated that they represent new members of the CYP71 gene family of plant cytochrome P450s. Clone pBSK3C7 contains the full-length coding sequence of a cytochrome P450, whilst pBSK4C3 lacks ca. 6 codons at the 5' end. The cytochromes P450 encoded by these clones were designated CYP71A5 and CYP71A6 (pBSK3C7 and pBSK4C3, respectively). Southern blot analysis indicated that the corresponding genes were present as single copies in the genome of N. racemosa. Northern blot analysis showed that a gene homologous with CYP71A5 was expressed in the related species N. cataria, but no homologue of CYP71A6 was detected in this species. Expression of CYP71A5 in N. racemosa was maximal in flowers, tissues within the apical bud, and young expanded leaves. That of CYP71A6 was maximal in older leaves. Expression of CYP71A5 occurred exclusively in trichomes present on the leaf surfaces, in contrast to that of CYP71A6, which occurred predominantly within the leaf blade tissues.     

Isolation and functional analysis of cytochrome P450 CYP153A genes from various environments

The cytochrome P450 CYP153 family is thought to mediate the terminal hydroxylation reactions of n-alkanes. We isolated 16 new P450 CYP153A genes (central region) from various environments such as petroleum-contaminated soil and groundwater, as well as one from the n-alkane-degrading bacterium Alcanivorax borkumensis SK2 (designated P450balk). The sequences of the new P450 genes were extended by PCR to generate full-length chimeric P450 genes, using the N- and C-terminal domains of P450balk. A differential CO-reduced P450 spectral analysis indicated that 8 P450 genes among the 16 chimeric genes were expressed in Escherichia coli to generate a soluble and functional enzyme. The several functional chimeric P450s and P450balk were further fused to the reductase domain of the self-sufficient P450 monooxygenase (P450RhF) at the C-terminus. E. coli cells expressing these self-sufficient P450 chimeric genes converted n-alkanes, cyclohexane, 1-octene, n-butylbenzene, and 4-phenyl-1-butene into 1-alkanols, cyclohexanol, 1,2-epoxyoctane, 1-phenyl-4-butanol, and 2-phenethyl-oxirane, respectively.     

cytochrome p450 superfamily in Arabidopsis thaliana: isolation of cDNAs,Differential Expression,and RFLP mapping of multiple cytochromes P450

We have isolated multiple cDNAs encoding cytochromes P450 (P450s) from Arabidopsis thaliana employing a PCR strategy. Degenerate oligonucleotide primers were designed from amino acid sequences conserved between two plant P450s, CYP71A1 and CYP73A2, including the heme-binding site and the proline-rich motif found in the N-terminal region, and 11 putative P450 fragments were amplified from first-strand cDNA from 7-day-old Arabidopsis as a template. With these PCR fragments as hybridization probes, 13 full-length and 3 partial cDNAs encoding different P450s have been isolated from an Arabidopsis cDNA library. These P450s have been assigned to either one of the established subfamilies: CYP71B, CYP73A, and CYP83A; or novel subfamilies: CYP76C, CYP83B, and CYP91A. The primary protein structures predicted from the cDNA sequences revealed that the regions around both the heme-binding site and the proline-rich motif were highly conserved among all these P450s. The N-terminal structures of the predicted P450 proteins suggested that these Arabidopsis P450s were located at the endoplasmic reticulum membrane. The loci of four P450 genes were determined by RFLP mapping. One of the clones, CYP71B2, was located at a position very close to the ga4 and gai mutations. RNA blot analysis showed expression patterns unique to each of the P450s in terms of tissue specificity and responsiveness to wounding and light/dark cycle, implicating involvement of these P450s in diverse metabolic processes.     

苹果蠹蛾细胞色素P450基因CYP332A19和CYP337B19的克隆及表达分析

【目的】本研究旨在通过克隆苹果蠹蛾Cydia pomonella细胞色素P450基因CYP332A19和CYP337B19,并对其进行序列和表达分析,以更好地了解这两个P450基因在植物次生物质解毒方面的作用,为进一步的功能研究提供依据。【方法】采用本地BLAST搜索苹果蠹蛾转录组数据库获得细胞色素P450基因cDNA序列,采用RT-PCR技术克隆目的基因的编码区。利用生物信息学软件分析目的基因的序列特征及与其他近缘物种的P450基因的系统进化关系。采用RT-qPCR技术测定目的基因在苹果蠹蛾不同发育阶段(卵、1-5龄幼虫、蛹和成虫)、4龄幼虫不同组织(头部、表皮、脂肪体、中肠和马氏管)以及4龄幼虫分别取食添加0.1%香豆素和0.5%槲皮素的人工饲料2 d后的表达水平。【结果】克隆获得苹果蠹蛾细胞色素P450基因CYP332A19(GenBank登录号: MF574708)和CYP337B19(GenBank登录号: MF574697)的全长cDNA序列,开放阅读框(ORF)分别长1 518和1 491 bp,分别编码505和496个氨基酸,其蛋白质分子量分别为58.586和57.734 kD,理论等电点分别为8.99和7.61。结构域分析显示,CYP332A19和CYP337B19中均包含包括血色素结合区在内的5个保守的细胞色素P450结构域。系统发育树显示,苹果蠹蛾CYP332A19与苹淡褐卷蛾Epighyas postvittana CYP332A9等CYP332A基因聚在一枝,而CYP337B19与稻纵卷叶螟Cnaphalocrocis medinalis CYP337B12和六星灯蛾Zygaena filipendulae CYP337B11等CYP337B基因聚在另一枝。RT-qPCR分析结果表明,CYP332A19和CYP337B19在苹果蠹蛾幼虫期的表达水平高于卵期的,分别在4龄幼虫脂肪体和中肠中的表达量最高。取食分别含0.1%香豆素和0.5%槲皮素的人工饲料2 d后,4龄幼虫体内的CYP332A19和CYP337B19相对表达量显著高于对照组(取食含2%DMSO的人工饲料)。【结论】CYP332A19和CYP332B19分别在苹果蠹蛾幼虫脂肪体和中肠中高表达,且在取食含香豆素和槲皮素的人工饲料的苹果蠹蛾幼虫体内表达量升高,说明这两个基因可能参与苹果蠹蛾对外源物质的解毒代谢过程。本研究的结果有助于我们了解苹果蠹蛾对寄主次生物质解毒代谢机理,为苹果蠹蛾防治提供新思路。