Molecular cloning and characterization of soybean peroxidase gene families

Plant peroxidases play major roles in many physiological processes. A soybean seedbud (21 days after flowering) Uni-ZAP XR cDNA library was screened with a peroxidase-specific probe. The probe was generated by 3′ rapid amplification of cDNA ends with soybean seedbud total RNA and a degenerate primer derived from a plant peroxidase conserved amino acid region (distal heme ligand). Positive clones were recovered by PCR using the degenerate peroxidase-specific primer and the vector primer T₇ flanking the cloning site. Four cDNAs, designated GmEpa1, GmEpa2, GmEpb1, and GmEpb2, contained 1298, 1326, 1171, and 1145 nucleotides, excluding poly(A) tail, and encoded mature proteins of 303, 303, 292, and 292 amino acids, respectively. The four predicted amino acid sequences showed homology to other peroxidases. GmEpa1 and GmEpa2 exhibited 97% amino acid identity, GmEpb1 and GmEpb2 exhibited 93% amino acid identity, and GmEpa1 and GmEpb1 exhibited 47% amino acid identity. GmEPa1 and GmEPb1 were expressed as fusion proteins in Escherichia coli. The recombinant fusion proteins were sequestered in inclusion bodies and active forms of the two denatured proteins were recovered after in vitro folding in a medium containing hemin, urea and Ca²⁺. GmEpa1 and GmEpa2 messages were detected in developing seed and root, while GmEpb1 and GmEpb2 messages were present in root, leaf, stem and seed pod. These cDNAs and cDNA-specific primers will allow investigations into peroxidase’s role in development, stress response and in other physiological processes.     

Cloning and characterization of a root sunflower peroxidase gene putatively involved in cell elongation

A cDNA-encoding a peroxidase (Helianthus annuus POX (HaPOX)1) was isolated and characterized from the roots of sunflower seedlings. This gene exhibited homology with other peroxidases from several higher-plant species, and its expression in the root growth was particularly abundant during cell expansion. To elucidate the precise functions of HaPOX1 in sunflower root, we examined its expression pattern in response to several plant growth regulators. Expression of HaPOX1 is down-regulated by abscisic acid (ABA), whereas indole-3-acetic acid (IAA) induced its expression. These results suggest that HaPOX1 expression is differentially regulated by phytohormonal components of signaling cascades. Since IAA appears to participate in the regulation of HaPOX1 expression, we postulate that the peroxidase encoded by HaPOX1 may be involved in the reactions that promote cell elongation during the early stage of root growth.     

Covalent immobilisation of manganese peroxidases (MnP) from Phanerochaete chrysosporium and Bjerkandera sp. BOS55

Manganese peroxidases (MnP) from Phanerochaete chrysosporium and Bjerkandera sp. BOS55 were immobilised in glutaraldehyde–agarose gels. Four different strategies were considered concerning the activation of the support (low or high density) and the ionic strength (low or high). In terms of immobilisation rate and yield, better results were obtained when low ionic strength conditions and high density activated support (75 μEq/ml) were used. Immobilisation proceeds initially with an ionic adsorption which facilitates the further covalent attachment of the enzyme to the support. An almost complete immobilisation has been attained in a very short period (0.5–2 h). Immobilisation maintained a high percentage of MnP activity for long periods of time (activity levels of 50–60% after more than 1 year at room temperature storage). Other desirable effects such as increased thermostability at 50–60 °C for MnP from Bjerkandera and higher resistance to high H₂O₂ concentrations for MnP for P. chrysosporium were also obtained. This latter is quite an interesting feature because it avoids the inactivation of the enzyme in the presence of an unbalanced concentration of H₂O₂. The improved characteristics of the immobilised MnP make its application in several fields such as the enzymatic oxidation of hardly degradable compounds more feasible.     

Functional characterization of an ammonium transporter gene from Lotus japonicus

NH₄⁺ is the main product of symbiotic nitrogen fixation and the external concentration of combined nitrogen plays a key regulatory role in all the different step of plant-rhizobia interaction. We report the cloning and characterization of the first member of the ammonium transporter family, LjAMT1;1 from a leguminous plant, Lotus japonicus. Sequence analysis reveals a close relationship to plant transporters of the AMT1 family. The wild type and two mutated versions of LjAMT1;1 were expressed and functionally characterized in yeast. LjAMT1;1 is transcribed in roots, leaves and nodules of L. japonicus plants grown under low nitrogen conditions, consistent with a role in uptake of NH₄⁺ by the plant cells.     

肺形侧耳变温结实相关基因Ppcsl-1的克隆及功能预测

CSL（CBF1/RBP-Jκ/suppressor of hairless/LAG-1）转录因子家族在真菌发育和细胞分化过程中扮演重要角色。前期研究已构建了肺形侧耳变温结实相关消减杂交文库,并从中筛选到一个代表csl基因部分序列的EST。通过TAIL PCR（thermal asymmetric interlaced PCR）技术克隆了该基因（Pleurotus pulmonarius csl-1,简写为 Ppcsl-1）,并利用RACE（rapid-amplification of cDNA ends）技术获得该基因的cDNA全长。Ppcsl-1 cDNA全长2 991bp,编码一个996个氨基酸组成的蛋白（命名为PpCSL-1）。进化分析显示在担子菌CSL中,PpCSL-1与糙皮侧耳Pleurotus ostreatus CSL（PoCSL）亲缘关系最近。荧光定量检测结果表明Ppcsl-1在菌丝经过5℃ 12h冷处理之后的表达量最高,表明其有可能被冷刺激诱导表达并在开启子实体形成的过程中起重要作用。     

Production, purification and partial enzymatic and molecular characterization of a laccase from the wood-rotting ascomycete Xylaria polymorpha

The hard wood-colonizing ascomycete Xylaria polymorpha, that is seemingly lacking peroxidases, produces laccase as sole ligninolytic oxidoreductase. The fungus secreted the enzyme preferably during the growth in complex media based on tomato juice. Addition of 2,5-xylidine considerably stimulated laccase production (up to 14,000 U l⁻¹). The enzyme was purified to homogeneity by anion exchange and size exclusion chromatography and characterized by biochemical and molecular methods. Xylaria laccase has a molecular mass of 67 kDa, a pI of 3.1 and an absorption maximum at 605 nm that is characteristic for blue copper proteins. It oxidized all typical laccase substrates including ABTS, 2,6-dimethoxyphenol, guaiacol as well as syringaldazine (catalytic efficiencies 3 × 10³ to 7 × 10⁴ M⁻¹ s⁻¹). The deduced amino acid sequence of one amplified laccase gene sequence between the copper binding regions 1 and 3 showed a high level of identity to some other laccases from ascomycetes. Furthermore, the sequence of an internal peptide fragment of the purified laccase was identical with an amino acid sequence deduced from the nucleotide sequence of the laccase gene. Xylaria laccase was found to oxidize a non-phenolic β-O-4 lignin model compound in presence of 1-hydroxybenzotriazole into the corresponding keto-form. The results of this study show that – in addition to ligninolytic basidiomycetes – also wood-dwelling ascomycetes can produce high titers of laccase that may be involved in the oxidation of lignin.     

Induction of lacI mutations in Big Blue Rat-2 cells treated with 1-(2-hydroxyethyl)-1-nitrosourea: a model system for the analysis of mutagenic potential of the hydroxyethyl adducts produced by 1,3-bis (2-chloroethyl)-1-nitrosourea.

We have investigated the genotoxic effects of 1-(2-hydroxyethyl)-1-nitrosourea (HENU). We have chosen this agent because of its demonstrated ability to produce N7-(2-hydroxyethyl) guanine (N7-HOEtG) and O⁶-(2-hydroxyethyl) 2′-deoxyguanosine (O⁶-HOEtdG); two of the DNA alkylation products produced by 1,3-bis (2-chloroethyl)-1-nitrosourea (BCNU). For these studies, we have used the Big Blue Rat-2 cell line that contains a lambda/lacI shuttle vector. Treatment of these cells with HENU produced a dose dependent increase in the levels of N7-HOEtG and O⁶-HOEtdG as quantified by HPLC with electrochemical detection. Treatment of Big Blue Rat-2 cells with either 0, 1 or 5 mM HENU resulted in mutation frequencies of 7.2±2.2×10⁻⁵, 45.2±2.9×10⁻⁵ and 120.3±24.4×10⁻⁵, respectively. Comparison of the mutation frequencies demonstrates that 1 and 5 mM HENU treatments have increased the mutation frequency by 6- and 16-fold, respectively. This increase in mutation frequency was statistically significant (P<0.001). Sequence analysis of HENU-induced mutations have revealed primarily G:C→A:T transitions (52%) and a significant number of A:T→T:A transversions (16%). We propose that the observed G:C→A:T transitions are produced by the DNA alkylation product O⁶-HOEtdG. These results suggest that the formation of O⁶-HOEtdG by BCNU treatment contributes to its observed mutagenic properties.     

环链棒束孢hsp90基因的克隆及冷热胁迫下的表达特征分析

通过本地Blast筛选转录组数据库方法,首次克隆了环链棒束孢热休克蛋白90基因全长cDNA序列,命名为Ichsp90（GenBank登录号KT944289）。克隆结果表明,该序列含有2 284个碱基,包括一个含2 097个碱基的开放阅读框,编码699个氨基酸,推测蛋白的分子量为79.23kDa,等电点（pI）为4.86,且含有5个Hsp90家族特征基序和胞质特征序列MEEVD,推导的氨基酸序列与其他丝状真菌相似性在92%-96%之间。用qRT-PCR方法分析了冷热胁迫下,该基因在环链棒束孢中的相对表达情况,结果表明：在4℃冷胁迫下15min检测到Ichsp90表达量下降到最低点,为对照的-1.8倍;随后表达量开始上升,至120min表达量是对照的1.07倍。在39℃高温胁迫下,60min Ichsp90表达量达到最高峰,为对照样品的5.02倍;随后表达量开始下降,至110min为对照样品的2.46倍。因此推测,Ichsp90基因在环链棒束孢抵抗外界温度胁迫中发挥重要的作用。     

Rapid determination of multi-locus sequence types of Listeria monocytogenes by microtemperature-gradient gel electrophoresis

This report presents a new method for identifying multi-locus sequence types of Listeria monocytogenes by microtemperature-gradient gel electrophoresis (μ-TGGE). Genomic comparison of L. monocytogenes serovar 1/2a strains EGD-e and F6854 allowed selection of novel polymorphic sequences lmo0386 and lmo0428 as optimum regions for μ-TGGE analysis, in addition to the previously identified lmo0297 gene. Sequence analysis of a total of 48 standard strains revealed that the strains could be grouped into 7 (lmo0386), 8 (lmo0428) and 12 (lmo0297) sequence types. The PCR products from 2, 4 and 4 sequence types of the lmo0386, lmo0428 and lmo0297 genes were selected as marker alleles, and μ-TGGE analysis of the mixture revealed adequate band separation on a single gel. Furthermore, the primer sets could be successfully mixed in a single tube for multiplex PCR, yielding a rapid and easy strategy for sequence type identification. For practical application, multiplex PCR was performed with Cy3-labeled primers against a sequence type-unknown sample isolated from meat. The resulting products were mixed with Cy5-labeled products of marker alleles whose sequence types were known, and μ-TGGE analysis was performed. Overlapping Cy3 and Cy5 patterns allowed identification of the sequence types at all 3 loci on a single gel. Moreover, the μ-TGGE analysis step took only 9 min. Thus, this novel method of multiplex PCR followed by μ-TGGE analysis could prove useful as a rapid and discriminative tool for tracing the strain types, contamination routes and sources of L. monocytogenes.     

Clustered genes within the genome of Arabidopsis thaliana encoding pectin methylesterase-like enzymes

We focused our attention on the isolation and regulation of genes encoding for pectin methylesterase (PME) isoenzymes in Arabidopsis thaliana (At). The present data reports the identification of two PME-like genes, named AtPME1 and AtPME2, that are closely linked in the At genome. These genes possess different structural organisations. While all higher plant PME characterised so far possess an intron at a similar location relative to their coding sequence, AtPME2 shows an additional intron whose presence within other higher plant PME-like genes has not been previously reported. Sequence comparison of the N-terminal region suggests that the secretory pathways of the putative AtPME1 and AtPME2 isoforms are different, and that this region may contribute to specify a biological function to each isoform. Moreover, phylogenetic analysis reflects the possible existence of functional groups of PME isoforms in higher plant species that seem to have been conserved during evolution.     

薰衣草叶片对低温胁迫的生理与分子响应机制

薰衣草(Lavandula angustifolia)作为名贵的芳香植物, 其生长、繁育、品质和产量均受低温影响。前期研究已获得1个耐低温薰衣草品种。该研究将对其处理的温度从20°C降至0°C, 揭示薰衣草响应冷胁迫的生理及分子调控机制, 同时结合薰衣草的细胞质膜透性、可溶性糖和蛋白质含量及抗氧化酶活性等生理变化。采用转录组学和生物信息学方法挖掘分析相关耐寒基因, 并探讨外施水杨酸缓解-10°C冻胁迫的可行性。研究发现7个编码脂肪酸去饱和酶和转移酶的基因(LaFADs)、3个参与合成可溶性糖的基因(LaBAM1和LaSS2)、19个编码胚胎晚期丰富蛋白的基因(LaLEAs)及7个编码过氧化物酶的基因(LaPODs), 这些基因在低温胁迫下均上调表达, 指导薰衣草合成并积累保护物质, 维持膜稳定性以应对胁迫。此外, 150 mg·L^-1水杨酸预处理能有效缓解植株冻害, 可作为低温保护剂。该研究丰富了薰衣草重要抗逆基因家族的遗传背景, 为后续分子遗传学功能分析和定向品种改良奠定基础。     

Transformation of 2,4,6-Trinitrotoluene (TNT) Reduction Products by Lignin Peroxidase (H8) from the White-Rot Basidiomycete Phanerochaete chrysosporium

White-rot fungi are known to degrade a wide range of xenobiotic environmental pollutants, including the nitroaromatic explosive 2,4,6-trinitrotoluene (TNT). TNT is first reduced by the fungal mycelium to aminodinitrotoluenes and diaminonitrotoluenes. In a second phase, reduced TNT metabolites are oxidatively transformed and mineralized. The extracellular oxidative enzyme of the ligninolytic system of these fungi includes the lignin peroxidases (LiP) and the manganese-dependent peroxidases (MnP). In the present study, we have shown that a cell-free enzymatic system containing fast protein liquid chromatography (FPLC)-purified LiP (H8) from the white-rot fungus Phanerochaete chrysosporium was able to completely transform 50 mg/L of 2,4-diamino-6-nitrotoluene (2,4-DA-6-NT) and 2-amino-4,6-dinitrotoluene (2-A-4,6-DNT) in 1 and 48 h, respectively. Veratryl alcohol (VA), often described as a mediator in the LiP-catalyzed oxidative depolymerization of lignin, was not required for the enzymatic transformation of 2,4-DA-6-NT or 2-A-4,6-DNT. 2,4-DA-6-NT was also shown to be a competitive inhibitor of the LiP activity measured through the oxidation of VA. Experiments using ¹⁴C-U-ring labeled compounds showed that 2-A-4,6-DNT was converted to 2,2'-azoxy-4,4' ,6,6'-tetranitrotoluene. No significant mineralization, measured by the release of ¹⁴CO2, was observed over 5 d.     

Expression profiles of two novel lipoxygenase genes in Populus deltoides

In the present study, we cloned two lipoxygenase genes, PdLOX1 and PdLOX2 (GenBank accession no. DQ131178, DQ131179), from Populus deltoides cv. ‘Lux’ (I-69/55). A prokaryotic expression analysis of PdLOX1 and PdLOX2 revealed that the encoded exogenous proteins were identical to the predicted molecular weights and possessed the expected lipoxygenase activities. Chromatogram analysis indicated that the two lipoxygenase mainly possess 13-LOX activity. Phylogenetic analysis of the derived amino acid sequences of known lipoxygenases revealed that PdLOX1 and PdLOX2 were members of the type 2 13-LOX family of genes. This class of lipoxygenases is known to be involved in biotic and abiotic stress. Using real-time RT-PCR, we evaluated PdLOX1 and PdLOX2 expression following exposure to a Poplar fungal pathogen (Marssonina brunnea f. sp. Multigermtubi), mechanical injury, methyl jasmonate (MeJA), or salicylic acid (SA). We report that both PdLOX1 and PdLOX2 expression levels were increased following exposure to M. brunnea f. sp. Multigermtubi, with the pathogen exerting a relatively stronger influence on PdLOX1 expression. Furthermore, expression levels of the two genes were also up-regulated by mechanical damage and exposure to MeJA. In contrast, both PdLOX1 and PdLOX2 expression was down-regulated by SA treatment. We propose that the two novel lipoxygenases may play an important role in Poplar resistance to biotic and abiotic stress.     

Cloning and characterization of a novel upstream untranslated exon of the mouse Fgf-1 gene

Fibroblast growth factor 1 (FGF-1 or aFGF), is the prototype member of the heparin-binding growth factors which are capable of angiogenesis in vivo. FGF-1 has been implicated in atherosclerosis, cancer, wound repair and inflammatory autoimmune diseases. As part of an effort to understand the role of FGF-1 in the etiopathogenesis of inflammation and cancer, we have undertaken steps to isolate and characterize the mouse Fgf-1 gene. Southern blotting and sequence analysis displayed considerable conservation within the coding and upstream untranslated regions of Fgf-1 in human, mouse, hamster, rat and bovine. By using primers derived from the 5′-untranslated exon of a rat prostate-specific Fgf-1 cDNA, a 220-bp product was amplified from mouse genomic DNA via PCR. Sequence analysis of this amplicon showed that there was 80% similarity with the corresponding region of the rat FGF-cDNA sequence. Primers designed from this amplicon and the Fgf-1 coding region were used to isolate multiple overlapping genomic clones spanning the entire mouse Fgf-1 gene. Sequencing analysis of the genomic sequence upstream from this novel 5'-untranslated exon did not reveal typical TATA, CCAAT sequences. It appears that the occurrence of multiple untranslated exons for FGF-1 is a highly conserved theme for this gene across species.     

新生隐球菌RBP1基因克隆与功能分析

新生隐球菌是自然界广泛存在的具荚膜的酵母型病原真菌,能侵染人类中枢神经系统引起真菌性脑膜炎,每年导致全球大约18万人死亡。本研究在前期隐球菌交配表达谱的基础上,选择一上调表达的RNA结合蛋白基因（CNAG_04772）,进行克隆和功能分析。结果表明该基因全长2 247bp,cDNA全长1 518bp,编码505个氨基酸组成的蛋白,含有2个RNA识别基序RRM1和RRM2,命名为RBP1。基因表达模式分析表明RBP1在隐球菌酵母细胞、担子以及担孢子阶段都有表达,交配菌丝阶段不表达;亚细胞定位分析表明Rbp1蛋白定位于隐球菌的细胞核和细胞质中。与野生型菌株H99相比,敲除突变体菌株能够交配并产生双核菌丝,但丧失产生担孢子的能力,而互补菌株与野生型菌株H99间无显著差异。致病力测定结果显示,敲除突变体菌株致病性显著降低。     

A fourth gene from the Candida albicans CDR family of ABC transporters

Using primers derived from a region of the Candida albicans CDR1 (Candida drug resistance) gene that is conserved in other ABC (ATP-binding cassette) transporters, a DNA fragment from a previously unknown CDR gene was obtained by polymerase chain reaction (PCR). After screening a C. albicans genomic library with this fragment as a probe, the complete CDR4 gene was isolated and sequenced. CDR4 codes for a putative ABC transporter of 1490 amino acids with a high degree of homology to Cdr1p, Cdr2p and Cdr3p from C. albicans (62, 59 and 57% amino acid sequence identity, respectively). Cdr4p has a predicted structure typical for cluster I.1 of yeast ABC transporters, characterized by two homologous halves, each comprising an N-terminal hydrophilic domain with consensus sequences for ATP binding and a C-terminal hydrophobic domain with six transmembrane helices. In contrast to the CDR1/CDR2 genes, the genetic structure of the CDR4 gene was conserved in 59 C. albicans isolates from six different patients. Northern hybridization analysis showed that the CDR4 gene was expressed in most isolates, but no correlation between CDR4 mRNA levels and the degree of fluconazole resistance of the isolates was found. In addition, a C. albicans mutant in which both copies of the CDR4 gene were disrupted by insertional mutagenesis was not hypersusceptible to fluconazole as compared to the parent strain. Unlike CDR1 and CDR2, CDR4 does not, therefore, seem to be involved in fluconazole resistance in C. albicans.     

Phenol degradation and colour removal in submerged culture of Pleurotus sajor-caju with paper mill effluents

The fungus Pleurotus sajor-caju secretes phenol-oxidases that enable the use of recalcitrant compounds as substrates. The residues of paper manufacture contain high lignin levels, which gives the effluents a characteristic brownish colour. To test the potential of P. sajor-caju cultures on reducing these parameters, we used 90% of raw effluents from medium consistency oxygen delignification and bleaching stages plus 10% of mineral solution and different levels of glucose (5-15 g L^-1) as substrate. We observed a greater fungal biomass in cultures using effluent than in controls. Cultures containing 10 to 15 g L^-1 of glucose resulted in about 42% colour reduction. The polyphenol content was also reduced by 58.9% by the 13th day of culture. In addition, we observed the secretion of laccases (211.44 U mL^-1 and 45.98 U mL^-1 using ABTS and syringaldazine, respectively) and peroxidases (6.11 U mL^-1-ABTS) both peaking at the 7th day of culture and with similar kinetics of production in different glucose concentrations.