Cloning and expression of peroxisomal Ascorbate Peroxidase gene from wheat

A full-length cDNA encoding wheat peroxisomal ascorbate peroxidase (pAPX) was cloned by Suppression Subtractive Hybridization (SSH) and in silico approach. The cDNA was 1027 bp in length and contained a complete ORF of 876 bp, which encodes a protein of 292 amino acid residues. Its deduced amino acids sequence had 84% identity with that of pAPX from barley. The gene was designated as Ta-pAPX. The Ta-pAPX homologous genes were mapped on wheat chromosome 7A and 7D using Chinese Spring nulli-tetrasomic lines analysis. Northern analysis indicated that, after inoculation by Erysiphe graminis Dc.f.sp. tritici, the expression of Ta-pAPX gene in Yangmai5 was enhanced, but its expression in wheat-Haynaldia villosa 6VS/6AL translocation lines changed a little. The results implied that Ta-pAPX may be related to susceptibility of wheat to powdery mildew. The complete coding sequence of Ta-pAPX was cloned into an expression vector pET32 (a+) and a protein with the same deduced molecular weight (MW) was expressed in E. coli BL21 (DE3), which showed ascorbate peroxidase activity.     

Ascorbate peroxidase – a hydrogen peroxide-scavenging enzyme in plants

Ascorbate peroxidase is a hydrogen peroxide-scavenging enzyme that is specific to plants and algae and is indispensable to protect chloroplasts and other cell constituents from damage by hydrogen peroxide and hydroxyl radicals produced from it. In this review, first, the participation of ascorbate peroxidase in the scavenging of hydrogen peroxide in chloroplasts is briefly described. Subsequently, the phylogenic distribution of ascorbate peroxidase in relation to other hydrogen peroxide-scavenging peroxidases using glutathione, NADH and cytochrome c is summarized. Chloroplastic and cytosolic isozymes of ascorbate peroxidase have been found, and show some differences in enzymatic properties. The basic properties of ascorbate peroxidases, however, are very different from those of the guaiacol peroxidases so far isolated from plant tissues. Amino acid sequence and other molecular properties indicate that ascorbate peroxidase resembles cytochrome c peroxidase from fungi rather than guaiacol peroxidase from plants, and it is proposed that the plant and yeast hydrogen peroxide-scavenging peroxidases have the same ancestor.     

In Silico Characterization and Homology Modeling of Thylakoidbound Ascorbate Peroxidase from a Drought Tolerant Wheat Cultivar

Ascorbate peroxidase,a haem protein (EC 1.11.1.11),efficiently scavenges hydrogen peroxide (H2O2) in cytosol and chloroplasts of plants.In this study,a fulllength coding sequence of thylakoid-bound ascorbate peroxidase cDNA (TatAPX) was cloned from a drought tolerant wheat cultivar C306.Homology modeling of the TatAPX protein was performed by using the template crystal structure of chloroplastic ascorbate peroxidase from tobacco plant (PDB: 1IYN).The model structure was further refined by molecular mechanic...     

The Ascorbate System in Two Bryophytes: Brachythecium velutinum and Marchantia polymorpha

The ascorbate system, one of the major antioxidant systems, has been studied in two bryophytes; a moss, Brachythecium velutinum (Hedw.) B., S. & G., and a liverwort, Marchantia polymorpha L. The moss and liverwort gametophytes contain ascorbate both in the reduced and oxidized form; utilize ascorbate in removing hydrogen peroxide by means of ascorbate peroxidase and reconvert to ascorbate its oxidation products by means of dehydroascorbate reductase and monodehydroascorbate reductase. Ascorbate oxidase activity was measured in the cytosolic fraction suggesting a localization of the enzyme different from more evolved organisms. The ascorbate content was maintained in the moss after drought stress while it declines in the liverwort, which seems more sensitive to water stress. Since ascorbate recycling is more efficient in the moss than in the liverwort, this seems to suggest a correlation between efficiency of ascorbate recycling and water stress tolerance. This revised version was published online in July 2006 with corrections to the Cover Date.     

对水稻第4号染色体长臂近端粒区一个过氧化物酶基因簇的结构分析

通过对定位BAC克隆q3037(H0207F01）的序列测定和分析,在其中一个22.5kb的区域发现一个由5个第三类过氧化物酶基因（依次命名为osp1、osp2、osp3、osp4、osp5）组成的基因簇,分析表明,osp1、osp2和osp3分别含1个内含子,osp4和osp5分别含2个内含子。该5个基因分别编码338、335、336、343和346个氨基酸残基的蛋白质而且都具有N端信号肽序列,其中OSP1、OSP4、OSP5为表离子过氧化物酶,OSP2、OSP3为阳离子过氧化物酶。对5个基因间的两两比较分析和进化分析结果表明：该基因簇是通过一系列的串联基因复制事件而形成;osp5与来自玉米的ap1和来自大麦（Hordeum vulgar)的prx7为潜在的直向同源基因,而且,osp1-5与ap1、prx7构成了分泌性植物过氧化物酶基因家族中的一个的分枝。     

Positive selection within sperm-egg adhesion domains of fertilin: an ADAM gene with a potential role in fertilization

Genes with a role in fertilization show a common pattern of rapid evolution. The role played by positive selection versus lack of selective constraints has been more difficult to establish. One problem arises from attempts to detect selection in an overall gene sequence analysis. I have analyzed the pattern of molecular evolution of fertilin, a gene coding for a heterodimeric sperm protein belonging to the ADAM (A disintegrin and A metalloprotease) gene family. A nonsynonymous to synonymous rate ratio (d(N)/d(S)) analysis for different protein domains of fertilin alpha and fertilin beta showed d(N)/d(S) < 1, suggesting that purifying selection has shaped fertilin's evolution. However, an analysis of the distribution of single positively selected codon sites using phylogentic analysis by maximum likelihood (PAML) showed sites within adhesion domains (disintegrin and cysteine-rich) of fertilin beta evolving under positive selection. The region 3' to the EGF-like domain of fertilin alpha, where the transmembrane and cytoplasmic tail regions are supposed to be localized, showed higher d(N) and d(S) than any other fertilin alpha region. However, it was not possible to identify positively selected codon sites due to ambiguous alignments of the carboxy-end region (ClustalX vs. DiAlign2). When this region was excluded from the PAML analysis, most single positively selected codon sites were concentrated within adhesion domains (cysteine-rich and EGF-like). The use of an ancestral sequence prior to a recent duplication event of fertilin alpha among non-Hominidae primates (Macaca, Papio, and Saguinus) revealed that the duplication is partially responsible for masking the detection of positively selected sites within the disintegrin domain. Finally, most ADAM genes with a potential role in sperm maturation and/or fertilization showed significantly higher d(N) estimates than other ADAM genes.     

GORDITA (AGL63) is a young paralog of the Arabidopsis thaliana Bsister MADS box gene ABS (TT16) that has undergone neofunctionalization

MIKC‐type MADS domain proteins are key regulators of flower development in angiosperms. B_sister genes constitute a clade with a close relationship to class B floral homeotic genes, and have been conserved for more than 300 million years. The loss‐of‐function phenotype of the A. thaliana B_sister gene ABS is mild: mutants show reduced seed coloration and defects in endothelium development. This study focuses on GORDITA (GOA, formerly known as AGL63), the most closely related paralog of ABS in A. thaliana, which is thought to act redundantly with ABS. Phylogenetic trees reveal that the duplication leading to ABS and GOA occurred during diversification of the Brassicaceae, and further analyses show that GOA has evolved under relaxed selection pressure. The knockdown phenotype of GOA suggests a role for this gene in fruit longitudinal growth, while over‐expression of GOA results in disorganized floral structure and addition of carpel‐like features to sepals. Given the phylogeny and function of other B_sister genes, our data suggest that GOA has evolved a new function as compared to ABS. Protein analysis reveals that the GOA‐specific ‘deviant’ domain is required for protein dimerization, in contrast to other MIKC‐type proteins that require the K domain for dimerization. Moreover, no shared protein interaction partners for ABS and GOA could be identified. Our experiments indicate that modification of a protein domain and a shift in expression pattern can lead to a novel gene function in a relatively short time, and highlight the molecular mechanism by which neofunctionalization following gene duplication can be achieved.     

Expression of the Stress-Related Genes for Glutathione S-Transferase and Ascorbate Peroxidase in the Most-Glycinin-Deficient Soybean Cultivar Tousan205 during Seed Maturation

Global analysis of gene expression profiles in most-glycinin-deficient cultivar Tousan205, was performed by DNA microarray analysis. It was confirmed that Tousan205 lacks mRNA expression of three glycinin subunit precursor genes, G1 (A1aB1x), G2 (A2B1a), and G5 (A3B4), and lacks G4 (A5A4B3) protein. Most glycinin subunits were deficient in mature seeds of Tousan205. We compared the gene expression of Tousan205 with those of parent cultivar, Tamahomare, which was used for crossbreeding of Tousan205. As a result, Tousan205 exhibited higher expression of some seed maturation proteins, and stress-related genes such as glutathione S-transferase and ascorbate peroxidase. This result indicates the possibility that the decrease of main storage protein, glycinin causes stress in soybean.     

Evaluation of whether accelerated protein evolution in chordates has occurred before, after, or simultaneously with gene duplication

Gene duplication and loss are predicted to be at least of the order of the substitution rate and are key contributors to the development of novel gene function and overall genome evolution. Although it has been established that proteins evolve more rapidly after gene duplication, we were interested in testing to what extent this reflects causation or association. Therefore, we investigated the rate of evolution prior to gene duplication in chordates. Two patterns emerged; firstly, branches, which are both preceded by a duplication and followed by a duplication, display an elevated rate of amino acid replacement. This is reflected in the ratio of nonsynonymous to synonymous substitution (mean nonsynonymous to synonymous nucleotide substitution rate ratio [Ka:Ks]) of 0.44 compared with branches preceded by and followed by a speciation (mean Ka:Ks of 0.23). The observed patterns suggest that there can be simultaneous alteration in the selection pressures on both gene duplication and amino acid replacement, which may be consistent with co-occurring increases in positive selection, or alternatively with concurrent relaxation of purifying selection. The pattern is largely, but perhaps not completely, explained by the existence of certain families that have elevated rates of both gene duplication and amino acid replacement. Secondly, we observed accelerated amino acid replacement prior to duplication (mean Ka:Ks for postspeciation preduplication branches was 0.27). In some cases, this could reflect adaptive changes in protein function precipitating a gene duplication event. In conclusion, the circumstances surrounding the birth of new proteins may frequently involve a simultaneous change in selection pressures on both gene-copy number and amino acid replacement. More precise modeling of the relative importance of preduplication, postduplication, and simultaneous amino acid replacement will require larger and denser genomic data sets from multiple species, allowing simultaneous estimation of lineage-specific fluctuations in mutation rates and adaptive constraints.     

黄芩过氧化物酶同工酶电泳和抗坏血酸过氧化物酶活性分析

对黄芩（Scutellaria baicalensis Georgi)二倍体和同源四倍体过氧化物酶进行了同工酶电泳分析及抗坏血酸过氧化物酶活性测定。结果表明,黄芩二倍体与同源四倍体过氧化物酶同工酶酶谱一致,但后者的着色程度大于前者,二年生黄芩叶子在快速区Rf为0．494和0．512处出现新的谱带,不同发育阶段和不同组织器官的谱带存在明显差异;根和叶中谱带数最多,花其次,种子最少;试管苗谱带数先减少后增加,并在整个培养过程中出现特征性谱带C。各组织器官抗坏血酸过氧化物酶总活力差异明显,顶芽最高,叶子次之,花最低。黄芩一年生和二年生各个多倍体株系叶子抗坏血酸过氧酶总活力均高于二倍体叶子抗坏血酸过氧化酶总活力。试管苗生长过程中抗坏血酶过氧化物酶活活力的变化与生长趋势一致,表明该酶与植株生长发育紧密相关。     

Recent gene conversions between duplicated glutamate decarboxylase genes (gadA and gadB) in pathogenic Escherichia coli

Escherichia coli have evolved adaptive systems to resist strongly acidic habitats in part through the production of 2 biochemically identical isoforms of glutamate decarboxylase (GAD), encoded by the gadA and gadB genes. These genes occur in E. coli and other members of the genospecies (e.g., Shigella spp.) and originated as part of a genomic fitness island acquired early in Escherichia evolution. The present duplicated gad loci are widely spaced on the E. coli chromosome, and the 2 genes are 97% similar in sequence. Comparison of the nucleotide sequences of the gadA and gadB in 16 strains of pathogenic E. coli revealed 3.8% and 5.0% polymorphism in the 2 genes, respectively. Alignment of the homologous genes identified a total of 120 variable sites, including 21 fixed nucleotide differences between the loci within the first 82 codons of the genes. Twenty-three phylogenetically informative sites were polymorphic for the same nucleotides in both genes suggesting recent gene conversions or intergenic recombination. Phylogenetic analysis based on the synonymous substitutions per synonymous site indicated 2 cases in which specific gadA and gadB alleles were more closely related to one another than to other alleles at the corresponding locus. The results indicate that at least 3 gene conversion events have occurred after the gad gene duplication in the evolution of E. coli. Despite multiple gene conversion events, the upstream regulatory regions and the 5' end of each gene remains distinct, suggesting that maintaining functionally different gad genes is important in this acid-resistance mechanism in pathogenic E. coli.     

Sequence and tissue-specific expression of a putative peroxidase gene from wheat (Triticum aestivum L.)

We have used a cDNA clone encoding a pathogen-induced putative wheat peroxidase to screen a genomic libary of wheat (Triticum aestivum L. cv. Cheyenne) and isolated one positive clone, lambda POX1. Sequence analysis revealed that this clone contains a gene encoding a putative peroxidase with a calculated pI of 8.1 which exhibits 58% and 83% sequence identity to the amino acid sequence of the turnip (Brassica rapa) peroxidase and a pathogen-induced putative wheat peroxidase, respectively. The two introns in the wheat gene are at the same positions as introns in the peroxidase genes of tomato and horseradish. Results of S1-mapping experiments suggest that this gene is neither pathogen-nor wound-induced in leaves but is constitutively expressed in roots.     

基因倍增研究进展

基因倍增是指DNA片段在基因组中复制出一个或更多的拷贝,这种DNA片段可以是一小段基因组序列、整条染色体,甚至是整个基因组。基因倍增是基因组进化最主要的驱动力之一,是产生具有新功能的基因和进化出新物种的主要原因之一。本文综述了脊椎动物、模式植物和酵母在进化过程中基因倍增研究领域的最新进展,并讨论了基因倍增研究的发展方向。     

单拷贝核基因在昆虫分子系统学中的应用

单拷贝核基因(single-copy nuclear gene,scnDNA)是指基因组中拷贝数目少,只有1个或几个拷贝的核基因,大多数是属于生物体内组成型表达的持家基因。单拷贝核基因是分子系统学中的一种极有价值的分子标记,在构建生命树的主干及主干和末梢之间的中间分枝中将起极其重要的作用。文章主要介绍了单拷贝核基因在昆虫分子系统学中的应用情况、一些单拷贝核基因的特点以及应用单拷贝核基因研究昆虫分子系统学时存在的问题及注意事项。     

过量表达水稻细胞质型OsAPXs基因提高转基因烟草的耐盐性

为了验证水稻(Oryza sativa L.)细胞质型APXs与细胞耐盐性的关系,实验分别将OsAPXa和OsAPXb（基因登录号：D45423、AB053297）转化到烟草(Nictiana tabacum,N.plum)植株中。Southern结果表明,二基因分别整合到烟草的基因组;Northern分析表明,外源基因在转基因烟草中得到高效表达;在碳酸盐逆境下,T2代转基因植株与野生型对照相比,其APX活性呈现显著的提高,T₂代品系的H₂O₂含量和叶片受害程度显著低于野生型;T₂代品系分别在含有10 mmol·L^-1 NaHCO₃、5 mmol·L^-1 Na₂CO₃的MS培养基上生长,根的生长受到抑制,叶片产生黄化;野生型烟草则难以存活。水稻细胞质型OsAPXs基因的过量表达提高了转基因烟草的耐盐性,揭示出OsAPXa和OsAPXb在碳酸盐逆境应答过程中发挥着重要的作用。     

The sex-peptide gene (Acp70A) is duplicated in Drosophila subobscura

A 3.1-kb region of Drosophila subobscura homologous to the Acp70A region of D. melanogaster, which contains the sex-peptide gene, was cloned and sequenced. This region contains an approximately 600-bp duplication that includes the sex-peptide and its 5′ and 3′ flanking regions. The preproteins are 54 and 56 amino acids long, respectively (as compared to 55 amino acids in D. melanogaster), and each includes a 19-amino-acid-long signal peptide. The C-terminal part of the mature peptide is highly conserved between D. melanogaster and the two copies of D. subobscura. In this species, both copies of the gene are transcribed and, like in D. melanogaster, only expressed in males. The duplicated region includes 300 bp upstream of the gene that would therefore seem sufficient for their expression in males. This region presents at its 5′ end a stretch 93-bp that has a high similarity with the corresponding region of D. melanogaster and could be part of a still unidentified regulatory element of these genes.