Molecular characterization and chromosomal localization of cytochrome P450 genes involved in the biosynthesis of cyclic hydroxamic acids in hexaploid wheat

The cyclic hydroxamic acids, 2,4-dihydroxy-1,4-benzoxazin-3-one (DIBOA) and 2,4-dihydroxy-7-methoxy-1,4-benzoxazin-3-one (DIMBOA), are defensive secondary metabolites found in gramineous plants including wheat, maize and rye. cDNAs for five cytochromes P450 (P450s) involved in DIBOA biosynthesis (CYP71C6, CYP71C7v2, CYP71C8v2, CYP71C9v1 and CYP71C9v2) were isolated from seedlings of hexaploid wheat [( Triticum aestivum L. cv. Chinese Spring (2n=6x=42, genomes AABBDD)] by RT-PCR and screening of a cDNA library. CYP71C9v1 and CYP71C9v2 are 97% identical to each other in amino acid and nucleotide sequences. The cloned P450 species showed 76-79% identity at the amino acid level to the corresponding maize P450 species CYP71C1-C4, which are also required for DIBOA biosynthesis. The wheat P450 cDNAs were heterologously expressed in the yeast ( Saccharomyces cerevisiae) strain AH22. Microsome fractions from yeast cells expressing these P450 species catalyzed the same reactions as their maize orthologs. The chromosomes carrying the cyp71C6- C9v1 orthologs were identified by Southern hybridization using aneuploid lines of Chinese Spring wheat. The cyp71C9v1 orthologs were located on the chromosomes of wheat homoeologous group-4. The orthologs of the other P450 genes, cyp71C7v2, cyp71C6 and cyp71C8v2, were located on group-5 chromosomes. The same P450 genes were also present in the three ancestral diploid species of hexaploid wheat, T. monococcum (AA), Aegilops speltoides [BB (approximately SS)] and Ae. squarrosa (DD).     

Expression, purification, and characterization of Bacillus subtilis cytochromes P450 CYP102A2 and CYP102A3: flavocytochrome homologues of P450 BM3 from Bacillus megaterium

The cyp102A2 and cyp102A3 genes encoding the two Bacillus subtilis homologues (CYP102A2 and CYP102A3) of flavocytochrome P450 BM3 (CYP102A1) from Bacillus megaterium have been cloned, expressed in Escherichia coli, purified, and characterized spectroscopically and enzymologically. Both enzymes contain heme, flavin adenine dinucleotide (FAD) and flavin mononucleotide (FMN) cofactors and bind a variety of fatty acid molecules, as demonstrated by conversion of the low-spin resting form of the heme iron to the high-spin form induced by substrate-binding. CYP102A2 and CYP102A3 catalyze the fatty acid-dependent oxidation of reduced nicotinamide adenine dinucleotide phosphate (NADPH) and reduction of artificial electron acceptors at high rates. Binding of carbon monoxide to the reduced forms of both enzymes results in the shift of the heme Soret band to 450 nm, confirming the P450 nature of the enzymes. Reverse-phase high-performance liquid chromatography (HPLC) of products from the reaction of the enzymes with myristic acid demonstrates that both catalyze the subterminal hydroxylation of this substrate, though with different regioselectivity and catalytic rate. Both P450s 102A2 and 102A3 show kinetic and binding preferences for long-chain unsaturated and branched-chain fatty acids over saturated fatty acids, indicating that the former two molecule types may be the true substrates. P450s 102A2 and 102A3 exhibit differing substrate selectivity profiles from each other and from P450 BM3, indicating that they may fulfill subtly different cellular roles. Titration curves for binding and turnover kinetics of several fatty acid substrates with P450s 102A2 and 102A3 are better described by sigmoidal (rather than hyperbolic) functions, suggesting binding of more than one molecule of substrate to the P450s, or possibly cooperativity in substrate binding. Comparison of the amino acid sequences of the three flavocytochromes shows that several important amino acids in P450 BM3 are not conserved in the B. subtilis homologues, pointing to differences in the binding modes for the substrates that may explain the unusual sigmoidal kinetic and titration properties.     

Catalytic Reaction of Basidiomycete Lentinula edodes Cytochrome P450, Le.CYP1 Enzyme Produced in Yeast

The basidiomycete Lentinula edodes (Le.) cytochrome P450, Le.CYP1 was functionally expressed in Saccharomyces cerevisiae. The microsomal fraction containing Le.CYP1 was prepared from the recombinant yeast and the Le.CYP1 was analyzed. The 7-ethoxycoumarin and benzo(a)pyrene were found to be the substrates of Le.CYP1 enzyme. Le.CYP1 converted 7-ethoxycoumarin to 7-hydroxycoumarin.     

家蚕细胞色素P450基因Bmcyp6u1的克隆、序列分析与表达谱

细胞色素P450第6亚家族基因为昆虫所特有,与抗性相关。为了检测家蚕Bombyx mori cyp6u1基因是否与耐氟性相关,首先克隆了cyp6u1基因。采用生物信息学方法获得与黑腹果蝇Drosophila melanogaster cyp6u1基因同源的家蚕B. mori cyp6u1基因序列, 预测该序列的开放阅读框（ORF）为1 476 bp, 编码491个氨基酸, 推定的蛋白质分子质量为56.15 kD, 等电点为9.23。以家蚕5龄第3天幼虫精巢cDNA为模板, 设计特异引物PCR扩增出一条约1 500 bp的条带, 大小与家蚕cyp6u1序列的ORF预测值接近, 命名为Bmcyp6u1基因（GenBank登录号：HM130560）。同源性分析表明, Bmcyp6u1基因与蜜蜂Apis mellifera的同源基因cyp6AS13的相似性为56%, 与拟南芥Arabidopsis thaliana的cyp72A82的相似性为48%, 与人Homo sapiens的cyp3A7基因的相似性为50%。芯片数据分析表明, Bmcyp6u1基因在家蚕5龄第3天幼虫各组织表达量很低, 只在精巢组织(5龄第3天)稍有表达, 推测该基因具有组织特异性。     

赤点石斑鱼两种芳香化酶cDNA的克隆及其表达的组织特异性

以赤点石斑鱼 (Epinephelusakaara)脑垂体中提取的RNA为模板 ,根据芳香化酶的保守序列设计引物 ,利用GeneRacerTM 技术 ,克隆出两种芳香化酶即脑芳香化酶 (P4 5 0aromB)和性腺芳香化酶 (P4 5 0aromA)的cDNA ,其全长分别为 190 1bp (编码 5 0 9aa)和 1833bp (编码 5 18aa)。序列分析结果表明 ,赤点石斑鱼两种芳香化酶cDNA序列的同源性为 5 1 6 % ,氨基酸序列之间同源性为 6 2 5 % ,与斜带石斑鱼两种芳香化酶氨基酸同源性分别为 94 7%和 97 9%。对 8个科的 10种鱼进行了分子系统进化树分析 ,结果与根据传统的形态学和生化特征分类进化地位基本一致。以特异性引物扩增雌、雄赤点石斑鱼各种组织 (垂体、嗅球、端脑、下丘脑、中脑、后脑、延脑、心脏、肾脏、肝脏、脾脏、性腺、鳃、胃、肠、皮肤、脂肪、肌肉、头肾、胸腺、鳔 ) ,以β actin作内标比较各组织芳香化酶基因表达量的差异 ,结果表明 ,赤点石斑鱼脑芳香化酶 (P4 5 0aromB)有广泛的组织分布 ,脑和垂体的表达量很高 ,各组织表达量有明显的雌、雄差异 ;而性腺芳香化酶 (P4 5 0aromA)表达主要集中于垂体和性腺 ,且不论雌雄 ,其性腺表达量均高于脑垂体 ,和P4 5 0aromB的表达模式明显不同 ,表现为在脑部 ,P4 5 0aromB表达量高于P4 5 0aromA ,而在性腺 ,     

绿盲蝽P450基因的克隆及增效醚对P450酶活性的抑制作用

为探讨P450介导的绿盲蝽Apolygus lucorum(Meyer-Dür)抗药性机制,合理使用杀虫药剂,本研究通过活体和离体抑制实验发现,增效醚(PBO)对绿盲蝽P450酶活性有显著的抑制作用:在处理时长为24h时,P450酶活性由未处理时的12.02pmol/min/mgPro.下降至1.63pmol/min/mgPro.,PBO对P450酶的抑制中浓度为0.256mmol/L。生物测定结果表明,PBO对三氟氯氰菊酯具有显著增效作用,增效7.2倍,而对吡虫啉、灭多威、马拉硫磷无显著增效作用。利用RT-PCR及RACE技术对绿盲蝽P450基因进行克隆,获得了2条CYP4家族基因,全长均为1631bp,含有完整的开放阅读框,编码501个氨基酸;序列比对表明这是一对等位基因,含有CYP4家族所有保守特征序列;同源性比较及系统发育分析显示这2个基因编码的氨基酸序列与褐飞虱Nilaparvata lugens CYP4CE1亲缘关系最近,同源性分别为41.5%和41.1%。     

Cloning and Sequencing of Cytochrome P450 1A Complementary DNA in Eel (Anguilla japonica)

Cytochrome P450 1A (CYP1A) complementary DNA was isolated from eel (Anguilla japonica) liver treated with 3-methylcholanthrene. The cDNA contained a 5′ untranslated region of 163 bp, an open reading flame of 1560 bp coding for 519 amino acids and a stop codon, and a 3′ untranslated region of 1730 bp. The predicted molecular weight was approximately 58.4 kDa. The deduced amino acid sequence exhibited identities with reported CYP1A sequences of 80% for rainbow trout, 79% for scup, 76% for plaice and butterfly fish, and 74% for toadfish. When compared with mammalian CYP proteins, the eel CYP1A was more similar to CYP1A1 (54%–56%) than to CYP1A2 (49%–52%). Northern and Southern blot analyses showed two distinct bands, suggesting the existence of another 3-methylcholanthrene-inducible CYP1A gene in eel. Received December 19, 1998; accepted February 18, 1999     

褐飞虱细胞色素P450单加氧酶基因CYP4CE1的克隆及表达谱

为了解P450基因在褐飞虱Nilaparvata lugens适应水稻品种过程中的重要作用,利用反转录聚合酶链式反应（RT-PCR）,快速扩增cDNA末端（RACE）和长距离聚合酶链式反应（LD-PCR）技术,克隆了褐飞虱4龄若虫的CYP4家族的一个P450单加氧酶基因,被命名为CYP4CE1。该基因的全长cDNA序列（2 160 bp）含有一个1 626 bp的开放阅读框（ORF）,编码541个氨基酸残基的蛋白质。通过GenBank数据库中的blastx搜索引擎进行同源性分析,结果表明CYP4CE1编码的蛋白与岸蟹 Carcinus maenas的CYP4C39（GenBank登录号：JC8026）的相似性最高,两者的氨基酸序列同源性达43％;其次与热带蟑螂 Blaberus discoidalis的CYP4C1（AAA27819）及黑腹果蝇Drosophila melanogaster的CYP4C3（NP_524598）的相似性也较高,氨基酸序列同源性分别达42％。氨基酸序列比对表明该蛋白含有CYP4家族成员的所有保守特征序列,如螺旋K（E--R--P）,氧结合结构域即螺旋I（AG--T）,血红素结合区（PF--G---C-G--F）以及CYP4成员的特有特征序列（EVDTFMFEGHDTT）等。使用Northern杂交检测CYP4CE1随时间的表达变化,结果表明：与饲养于感虫水稻台中1号（Taichung Native 1,TN1）上的若虫相比,在取食中度抗性水稻Minghui 63（MH63）秧苗12,24,48,72 h的各时间段的褐飞虱体内,该基因有2.1倍的过量表达且表达水平保持稳定。进一步通过Northern杂交检测该基因的组织表达特异性,结果显示：该基因在取食TN1秧苗的若虫脂肪体中表达量最高,在肠道组织及体壁中的表达水平较低;褐飞虱取食MH63秧苗24 h后,该基因在体壁及脂肪体中的表达量略有上升（各约1.2倍）,而在肠道组织中的表达量则大幅升高（约12倍）。肠道整体原位杂交表明,CYP4CE1在取食TN1秧苗的若虫的肠道组织及马氏管中均有本底水平的表达;若虫取食MH63秧苗后,该基因在上述肠道各区段表达水平明显增强。结果提示,在褐飞虱与水稻互作过程中CYP4CE1的重要功能之一可能是参与水稻有毒次生物质的代谢。     

棉铃虫细胞色素P450 cDNA片段的克隆与序列分析

以 5龄实验室敏感品系棉铃虫Helicoverpaarmigera的总RNA为模板 ,采用简并性引物 ,利用反转录 -多聚酶链式反应 (RT PCR)扩增出了 2个新的长度分别为 2 3 7bp和 2 40bp的cDNA片段。序列分析表明 ,2 3 7bp的cDNA片段与棉铃虫P45 0CYP4家族有较高的相似性 ,最高达 73 %;而 2 40bp的cDNA片段与CYP6家族有较高的相似性 ,最高达 49%。     

Multiple P450alk (cytochrome P450 alkane hydroxylase) genes from the halotolerant yeast Debaryomyces hansenii

The halotolerant alkane-assimilating yeast Debaryomyces hansenii was examined for P450 alkane hydroxylase genes known to be required for alkane assimilation in Candida. Four distinct P450alk gene segments and an allelic segment were isolated using PCR based on degenerate primers derived from the CYP52 family of alkane-inducible P450 genes. A screen of a genomic library (15–20 kb inserts) constructed for this study, using a probe based on the PCR-isolated segments, yielded seven clones. This has led to the isolation and sequence of two full-length genes DH-ALK1 and DH-ALK2. These genes, each with an ORF of 1557 bp (519 aa), contained no apparent introns and showed 64% nucleotide sequence homology (61% based on the deduced amino acid sequences). The deduced proteins had predicted molecular weights of 59,254 Da (DH-ALK1) and 59,614 Da (DH-ALK2) and have been designated CYP52A12 and CYP52A13 by the P450 Nomenclature Committee. Phylogenetic analysis based on Neighbor Joining Tree showed that DH-ALK1 and DH-ALK2 constitute new genes located on two distinct branches and are most related to the gene CYP52A3 (60% deduced aa homology) and are least related to the gene CYP52C2 (41% deduced aa homology), both of C. maltosa. The isolated genes will provide tools to better understand the diversity of the P450alk family in eukaryotic microorganisms adapted to varied environmental conditions.     

唐鱼细胞色素P450 1A基因cDNA克隆及分析

鱼类细胞色素P450 1A(CYP1A)基因作为一种有效的生物标志物已被广泛应用于环境污染物的评价,唐鱼在水环境污染监测中有较好的应用优势,为建立唐鱼在水环境的检测的有效生物标志物方法,本研究对其CYP1A基因进行克隆。利用RT-PCR法扩增出一条651bp的唐鱼CYP1A基因cDNA片段,该片段与其他物种的CYP1A基因具有很高的相似性。在此基础上进行5'端扩增和3'RACE获得了唐鱼CYP1A基因的全长cDNA序列,其长度为2177bp,其中编码区长1566bp,编码521个氨基酸残基组成的蛋白质,推测该蛋白质分子量大小为58.5kDa,理论等电点为5.65。所得序列具有CYP1A分子的主要特征和功能域,包括亚铁血红素结合区、酶功能所需的精氨酸密码子和终止密码子。推导出的氨基酸序列与斑马鱼、底鳉、虹鳟、大西洋鳕、人、鼠等脊椎动物同源性分别为87.7%、70.1%、76.2%、62.2%、54.5%、55.7%。