Molecular cloning and characterization of 1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate reductase (CaHDR) from Camptotheca acuminata and its functional identification in Escherichia coli

Camptothecin is an anti-cancer monoterpene indole alkaloid. The gene encoding 1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate reductase (designated as CaHDR), the last catalytic enzyme of the MEP pathway for terpenoid biosynthesis, was isolated from camptothecin-producing Camptotheca acuminata. The full-length cDNA of CaHDR was 1686 bp encoding 459 amino acids. Comparison of the cDNA and genomic DNA of CaHDR revealed that there was no intron in genomic CaHDR. Southern blot analysis indicated that CaHDR belonged to a low-copy gene family. RT-PCR analysis revealed that CaHDR expressed constitutively in all tested plant organs with the highest expression level in flowers, and the expression of CaHDR could be induced by 100 microM methyl-jasmonate (MeJA), but not by 100 mg/L salicylic acid (SA) in the callus of C. acuminata. The complementation of CaHDR in Escherichia coli ispH mutant MG1655 demonstrated its function.     

Molecular cloning, expression profiling and functional analysis of a DXR gene encoding 1-deoxy-D-xylulose 5-phosphate reductoisomerase from Camptotheca acuminata

As the second enzyme of the non-mevalonate terpenoid pathway for isopentenyl diphosphate biosynthesis, DXP reductoisomerase (DXR, EC: 1.1.1.267) catalyzes a committed step of the MEP pathway for camptothecin (CPT) biosynthesis. In order to understand more about the role of DXR involved in the CPT biosynthesis at the molecular level, the full-length DXR cDNA sequence (designated as CaDXR) was isolated and characterized for the first time from a medicinal Nyssaceae plant species, Camptotheca acuminata. The full-length cDNA of CaDXR was 1823 bp containing a 1416 bp open reading frame (ORF) encoding a polypeptide of 472 amino acids. Comparative and bioinformatic analyses revealed that CaDXR showed extensive homology with DXRs from other plant species and contained a conserved transit peptide for plastids, an extended Pro-rich region and a highly conserved NADPH binding motif in its N-terminal region owned by all plant DXRs. Phylogenetic analysis indicated that CaDXR was more ancient than other plant DXRs. Tissue expression pattern analysis revealed that CaDXR expressed strongly in stem, weak in leaf and root. CaDXR was found to be an elicitor-responsive gene, which could be induced by exogenous elicitor of methyl jasmonate. The functional color complementation assay indicated that CaDXR could accelerate the biosynthesis of carotenoids in the Escherichia coli transformant, demonstrating that DXP reductoisomerase plays an influential step in isoprenoid biosynthesis.     

Molecular cloning, characterization and expression of a jasmonate biosynthetic pathway gene encoding allene oxide cyclase from Camptotheca acuminata

AOC (allene oxide cyclase; EC 5.3.99.6), an essential enzyme in jasmonic acid and its methyl ester biosynthesis, was cloned from Camptotheca acuminata (named as CaAOC), a native medicinal plant species in China. CaAOC had significant similarity at the amino-acid level with AOCs from other plant species. Comparison between the sequences of the full-length cDNA and genomic DNA of CaAOC revealed that the genomic DNA of CaAOC contained an 89-bp intron and a 240-bp intron. Southern-blot analysis indicated that CaAOC was a multiple-copy gene, and real-time quantitative PCR analysis showed that CaAOC was expressed constitutively in all organs tested, with the highest expression level in leaves. The results from treatment experiments using different signalling components, including methyl jasmonate, abscisic acid, salicylic acid and H(2)O(2), revealed that expression of CaAOC had a prominent diversity. Heavy metal (copper) significantly enhanced CaAOC expression, whereas wounding (induced by UV-B) was not so effective.     

Cloning and characterization of a novel member of the cytochrome P450 subfamily IVA in rat prostate

To isolate cDNAs for forms of cytochrome P450 from rat prostate, a lambda gt11 cDNA library from this tissue was screened with a mixture of oligonucleotide probes directed against the conserved heme binding region of different P450 isozymes. A cDNA clone (PP1) encoding a part of a novel form of cytochrome P450 was isolated and the deduced amino acid sequence showed 76% identity with cytochrome P450 IVA1, indicating that PP1 is a member of the same subfamily. Northern blot analysis of total RNA from prostates of untreated rats revealed that two mRNAs of approximately 2.8 and 2.2 kb hybridize to PP1. The level of mRNA was induced fivefold above the level in intact animals by androgen treatment of castrated rats. Analysis of poly(A)+RNA levels in different tissues on Northern blots showed high constitutive expression of PP1 in the kidney, but no signal was detectable with RNA from liver; a weak signal was detected in the retina. Subsequent screening of a rat kidney cDNA library led to the isolation of the full-length clone KP1, which differs from Pp1 only in three nucleotide positions. KP1 is 1,957 bp long and contains a 1,527-bp-long open reading frame encoding a protein of 508 amino acids. In situ hybridization of rat kidney sections with PP1 showed that this P450 form is expressed in the outer stripe of the outer medulla, indicating its localization in the proximal tubules.     

A cDNA encoding a rat mitochondrial cytochrome P450 catalyzing both the 26-hydroxylation of cholesterol and 25-hydroxylation of vitamin D3: gonadotropic regulation of the cognate mRNA in ovaries

A cDNA expression library prepared from rat liver RNA was screened with a polyclonal antibody specific for mitochondrial vitamin D3 25-hydroxylase and a cDNA for rabbit liver mitochondrial cytochrome P450c26 (CYP 26), yielding cDNA clones with identical sequences. The deduced amino acid sequence derived from a 1.9-kb full-length cDNA was 73% identical to that of rabbit cytochrome P450c26. A monoclonal antibody was used to demonstrate that the product of the 1.9-kb cDNA clone was targeted to the mitochondrial compartment when expressed in COS cells. Mitochondrial membranes containing the expressed protein showed both vitamin D3 25-hydroxylase and cholesterol 26-hydroxylase activities when reconstituted with ferredoxin reductase and ferredoxin, demonstrating that the same P450, designated as P450c26/25, can catalyze both reactions. Northern blot analysis revealed that the P450c26/25 cDNA hybridizes with a 2.4-kb RNA from rat liver and unstimulated ovaries. Treatment of rats with pregnant mare's serum gonadotropin resulted in a fivefold increase in the 2.4-kb mRNA as well as the appearance of a 2.1-kb mRNA species in the ovaries. Our findings document the presence of a regulated bifunctional mitochondrial cytochrome P450 capable of catalyzing the 25-hydroxylation of vitamin D3 and the 26-hydroxylation of cholesterol.     

Molecular cloning of monkey liver cytochrome P-450 cDNAs: similarity of the primary sequences to human cytochromes P-450.

Three cDNAs coding for monkey cytochrome P-450 (P450) 2C, 2E and 3A (MKmp13, MKj1 and MKnf2, respectively) were isolated from a lambda gt11 cDNA library of a liver from a 3-methylcholanthrene (3MC)-treated crab-eating monkey, using cDNA fragments for human P450 2C, 2E and 3A as respective probes. MKmp13 and MKnf2 were 1901 and 2032 bp long, containing entire coding regions for polypeptides of 490 and 503 residues, respectively. The deduced N-terminal amino acid sequences of MKmp13 and MKnf2 were identical with those of P450-MK1 and P450-MK2, which had been purified from liver microsomes of untreated and polychlorinated biphenyl (PCB)-treated crab-eating monkeys, respectively. MKj1 was 1508 bp long, encoding a polypeptide of 449 residues, which is presumed to lack N-terminal 45 residues as compared with the sequence for human P450 2E1. Northern blot analysis indicated that monkey P450 2C, 2E and 3A mRNAs were expressed constitutively in monkey livers. P450 2E and 3A mRNAs were induced by both 3MC and PCB, while P450 2C mRNA was induced only by PCB. The deduced amino acid sequences of four monkey cytochrome P-450 cDNAs, including P450 1A1 (MKah1) which we isolated previously, were more than 92% identical with those of corresponding human cytochrome P-450 cDNAs.     

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.     

Mouse pulmonary cytochrome P-450 naphthalene hydroxylase: cDNA cloning, sequence, and expression in Saccharomyces cerevisiae.

We have isolated a cDNA clone, Nah-2, encoding the cytochrome P-450Nah (naphthalene hydroxylase) from a mouse lung lambda ZAP cDNA library using anti-cytochrome P-450Nah IgG as a probe. This same antibody selectively blocked [Nagata, K., Martin, B.M., Gillette, J.R., & Sasame, H.A. (1990) Drug Metab. Dispos. 18, 557-564] the cytochrome P-450 in mouse lung microsomes that catalyzed the conversion of naphthalene to (1R,2S)-naphthalene 1,2-oxide, which has been postulated as a causative agent in the naphthalene-induced tissue-specific necrosis of Clara cells in mouse lung. The toxic effect is seen in mouse and not in rat. The cDNA encodes a polypeptide of 491 amino acids with a molecular mass of 50 kDa. Northern blot analysis with an Nah-2-specific probe revealed that the mRNA is expressed in a species- and tissue-specific manner, present only in mouse lung and liver and not in that of rat. The mRNA encoding Nah-2 is constitutively expressed and is not induced by either phenobarbital, pyrazole, pregnenolone 16 alpha-carbonitrile, or 3-methylcholanthrene. Comparative amino acid sequence analyses with other documented members of the P-450 gene superfamily revealed that this encoded protein is in the IIF subfamily. To analyze its substrate specificity, the cDNA was inserted into the vector, pAAH5, and expressed in the Saccharomyces cerevisiae strain, AH22. The presence of cytochrome P-450Nah in the microsomes isolated from transformed cells and analyzed by Western blot was confirmed by immunocomplexing product with anti-cytochrome P450Nah IgG. Furthermore, activity toward naphthalene in the microsomes from the transformed cells established that this clone encodes a naphthalene hydroxylase.(ABSTRACT TRUNCATED AT 250 WORDS)     

High-level expression of functional human cytochrome P450 1A2 in Escherichia coli.

Enzymatically active human cytochrome P450 1A2 was expressed in Escherichia coli utilizing the pCWori+ vector containing a modified cDNA. The coding sequence for the NH2-terminal region of the protein was modified by the alignment and substitution of a 27 bp segment from a modified bovine P450 17A1 cDNA onto the 5' end of the open reading frame of P450 1A2 at amino acid 21. The expressed chimeric P450 was produced at a high level in a functionally intact form, as assayed by the formation in vivo of the 449 nm absorbance band of the CO complex of the reduced hemoprotein. E. coli membrane preparations were shown to contain P450 1A2, which was active in the 2-hydroxylation of estradiol, and the O-deethylation of 7-ethoxycoumarin and 7-ethoxyresorufin, when reconstituted with recombinant rat liver NADPH-cytochrome P450 reductase.     

Characterization of 5-enolpyruvylshikimate 3-phosphate synthase gene from Camptotheca acuminata

5-enolpyruvylshikimate 3-phosphate synthase (EPSPS; 3-phosphoshikimate 1-carboxyvinyl-transferase; EC 2.5.1.19) is a critical enzyme in the shikimate pathway. The full-length EPSPS cDNA sequence (CaEPSPS, GenBank accession number: AY639815) was cloned and characterized for the first time from woody plant, Camptotheca acuminata, using rapid amplification of cDNA ends (RACE) technique. The full-length cDNA of CaEPSPS was 1778 bp containing a 1557 bp ORF (open reading frame) encoding a polypeptide of 519 amino acids with a calculated molecular mass of 55.6 kDa and an isoelectric point of 8.22. Comparative and bioinformatic analyses revealed that CaEPSPS showed extensive homology with EPSPSs from other plant species. CaEPSPS contained two highly conserved motifs owned by plant and most bacteria EPSPSs in its N-terminal region. Phylogenetic analysis revealed that CaEPSPS belonged to dicotyledonous plant EPSPS group. Tissue expression pattern analysis indicated that CaEPSPS was constitutively expressed in leaves, stems and roots, with the lower expression being found in roots. The coding sequence of CaEPSPS gene was successfully subcloned in a plasmid-Escherichia coli system (pET-32a), and the cells containing the plasmid carrying the CaEPSPS gene exhibited enhanced tolerance to herbicide glyphosate, compared to the control.     

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 ,     

Olfactory-specific cytochrome P-450. cDNA cloning of a novel neuroepithelial enzyme possibly involved in chemoreception

We isolated cDNA clones for cytochrome P-450 genes expressed in the olfactory neuroepithelium by screening a corresponding rat cDNA library. Sequence analysis and RNA blot hybridization revealed a new cytochrome P-450, designated cytochrome P-450olf1, which is the first reported cytochrome P-450 mRNA uniquely expressed in the chemosensory organ. Cytochrome P-450olf1 shows intermediate level of sequence similarity (38-53% identity) to several liver cytochrome P-450 enzymes, suggesting that it belongs to the cytochrome P-450II family, but defines a new subfamily (cytochrome P-450IIG) within it. Cytochrome P-450II enzymes are known to process diverse organic compounds, including odorants. This, together with the specificity of cytochrome P-450olf1 to the sensory neuroepithelium, may indicate a role for this protein in olfactory reception.     

产喜树碱喜树内生真菌的筛选及喜树内生真菌的SRAP分析

生活在宿主植物里的内生真菌是很重要的药用资源。喜树是中国的传统药用植物。从喜树植物中分离得到了大约50种菌株,其中一株产喜树碱的菌株通过形态学鉴定为青霉属,这是首次在喜树植物中发现产喜树碱的青霉属菌株。为研究简单序列重复相关序列扩增多态性(SRAP)方法在喜树内生真菌中应用的可行性,选择了十株喜树内生真菌进行SRAP多态性分析。SRAP引物共扩增出1 295条带,而这些菌株也被分为三大类。这些结果表明,SRAP研究喜树内生真菌具有高效性,是讨论喜树内生真菌的遗传多样性的有效方法。     

Isolation of a cDNA and a genomic clone encoding cinnamate 4-hydroxylase from Arabidopsis and its expression manner in planta.

We have isolated a cDNA for a cytochrome P450, cinnamate 4-hydroxylase (C4H), of Arabidopsis thaliana using a C4H cDNA from mung been as a hybridization probe. The deduced amino acid sequence is 84.7% identical to that of mung bean C4H and therefore was designated CYP73A5. The CYP73A5 protein was expressed in insect cells using the baculovirus expression system and when reconstituted with lipid and NADPH-cytochrome P450 reductase resulted in C4H activity with a specific activity of 68 nmol min-1 nmol-1 P450. Southern blot analysis revealed that CYP73A5 is a single-copy gene in Arabidopsis. C4H (CYP73A5) expression was apparently coordinated in Arabidopsis with both PAL1 and 4CL in response to light and wounding. Although the light induction of CHS followed a time course similar to that observed with C4H, no induction of CHS was detected upon wounding. On the other hand, the C4H expression patterns exhibited no significant coordination with those of PAL2 and PAL3. A C4H promoter region of 907 bp contained all of the three cis-acting elements (boxes P, A, and L) conserved among the PAL and 4CL genes so far reported as controlling expression.