Molecular cloning and characterization of a glutathione S-transferase gene from Ginkgo biloba.

Glutathione S-transferases (GSTs) play an important role in the response of plants to changing environmental conditions. Here, we report the cloning of the GST gene for GST from Ginkgo biloba, a native medicinal plant species in China, by rapid amplification of cDNA ends (RACE). The full-length cDNA (designated as GbGST) was 1008 bp and contained a 684 bp open reading frame (ORF) encoding a polypeptide of 228 amino acids. The genomic sequence of GbGST was also obtained. Semi-quantitative RT-PCR analysis revealed that GbGST expressed in all tested tissues of G. biloba, including leaf, root and stem and the expression of GbGST could be induced by UV, MJ and drought treatments, suggesting that GbGST was potentially involved in plant's stress tolerance. To our knowledge, this is the first GST cDNA cloned from Ginkgoaceae. Based on comparative analyses of amino acid sequence, phylogeny, predicted three-dimensional structure together with the gene structure, the GbGST should be classified into the tau class.     

Whole genome survey of the glutathione transferase family in the brown algal model Ectocarpus siliculosus

We report here an exhaustive analysis of the glutathione transferases (GSTs) in the model brown alga Ectocarpus siliculosus using available genomic resources. A genome survey revealed the presence of twelve cytosolic GSTs, belonging to the Sigma class, two pseudogenes, one GST of the Kappa class, and three microsomal GSTs of the MGST3 family of membrane associated protein involved in eicosanoid and glutathione metabolism. Gene structure and phylogenetic analyses demonstrated the partition of the Sigma GSTs into two clusters which have probably evolved by duplication events. Gene expression profiling was conducted after the addition of high concentrations of chemicals, such as H(2)O(2), herbicides, heavy metals, as well as fatty acid derivatives, in order to induce stress conditions and to monitor early response mechanisms. The results of these experiments suggested that E. siliculosus GST genes are recruited in different and specific conditions. In addition, heterologous expression in yeast of two E. siliculosus microsomal GST showed that these enzymes feature peroxidase rather than transferase activity. The potential involvement of E. siliculosus GST in the metabolism of oxygenated polyunsaturated fatty acids is discussed.     

Purification of a phi-type glutathione S-transferase from pumpkin flowers,and molecular cloning of its cDNA

A major species of glutathione S-transferase (GST), Pugf, was highly purified from pumpkin flowers. Two-dimensional electrophoresis of the purified enzyme gave two adjacent protein spots. The specific activity of the purified enzyme was 2.4 micromol min(-1) mg(-1) protein for 1-chloro-2,4-dinitrobenzene. This value is one to two orders of magnitude lower than that of pumpkin tau-type GSTs. The expression pattern of Pugf in healthy pumpkin plants and responses to various stresses were examined by western blotting. Pugf was found in high concentrations in petioles, stems, and roots as well as flowers, and was more abundant in expanding young organs than in fully expanded mature organs. Dehydration caused a slight increase in its concentration, but high and low temperatures, salty stress, and 2,4-dichlorophenoxyacetic acid seemed to have no effects. A cDNA encoding Pugf was cloned and sequenced. Sequence comparison with other plant GSTs suggested that it should be classified as a phi-type GST.     

二斑叶螨抗螺螨酯品系GST基因的克隆与表达分析

【目的】揭示二斑叶螨Tetranychus urticae对螺螨酯的分子抗性机理。【方法】利用RT-PCR克隆了二斑叶螨的谷胱甘肽-S-转移酶（glutathione S-transferase, GST)基因 cDNA 全长序列, 采用生物信息学软件分析了克隆基因的编码蛋白特性; 利用实时荧光定量PCR 方法分析GST基因在二斑叶螨的螺螨酯抗性与敏感品系中的表达差异。【结果】克隆获得的谷胱甘肽-S-转移酶2个基因分别被命名为TuGSTd1和TuGSTd2 (GenBank登录号分别为： KC445659和KC445660)。序列分析发现, TuGSTd1的开放阅读框长度为648 bp, 编码215个氨基酸, 分子量约为24.47 kDa, 理论等电点为5.49; TuGSTd2的开放阅读框为648 bp, 编码215个氨基酸, 分子量约为24.57 kDa, 理论等电点为6.33。系统发育分析表明这两个基因与桔全爪螨Panonychus citri Delta家族的GST基因的氨基酸序列一致性为93%。实时荧光定量PCR 结果表明, TuGSTd1和TuGSTd2在二斑叶螨抗螺螨酯品系中的相对表达量分别为敏感品系的5.60和3.75 倍。【结论】 GST基因在二斑叶螨抗螺螨酯品系中的相对表达量均显著高于敏感品系, 据此推测GST基因的过量表达可能与其对螺螨酯的抗性形成有关。     

Molecular cloning and characterization of alpha-class glutathione S-transferase gene from the liver of silver carp, bighead carp, and other major Chinese freshwater fishes

Two full-length cDNAs encoding glutathione S-transferase (GST) were cloned and sequenced from the hepatopancreas of planktivorous silver carp (Hypophthalmichthys molitrix) and bighead carp (Aristichthys nobilis). The silver carp and bighead carp GST cDNA were 920 and 978 bp in length, respectively, and both contained an open reading frame that encoding 223 amino acids. Partial GST cDNA sequences were also obtained from the liver of grass carp (Ctenopharyngodon idellus), crucian carp (Carassius auratu), mud carp (Cirrhinus molitorella), and tilapia (Oreochromis nilotica). All these GSTs could be classified as alpha-class GSTs on the basis of their amino acid sequence identity with other species. The three-dimensional structure of the silver carp GST was predicted using a computer program, and was found to fit the classical two-domain GST structure. Using the genome walker method, a 875-bp 5'-flanking region of the silver carp GST gene was obtained, and several lipopolysaccharide (LPS) response elements were identified in the promoter region of the phytoplanktivorous fish GST gene, indicating that the GST gene expression of this fish might be regulated by LPS, released from the toxic blue-green algae producing microcystins. To compare the constitutive expression level of the liver GST gene among the six freshwater fishes with completely different tolerance to microcystins, beta-actin was used as control and the ratio GST/beta-actin mRNA (%) was determined as 130.7 +/- 6.6 (grass carp), 103.1 +/- 8.9 (bighead carp), 92.6 +/- 15.0 (crucian carp), 72.3 +/- 7.8 (mud carp), 58.8 +/- 11.5 (silver carp), and 33.6 +/- 13.7 (tilapia). The constitutive expression level of the liver GST gene clearly shows that all the six freshwater fishes had a negative relationship with their tolerance to microcystins: high-resistant fishes (phytoplanktivorous silver carp and tilapia) had the lowest tolerance to microcystins and the high-sensitive fish (herbivorous grass carp) had the highest tolerance to microcystins. Taken together with the reciprocal relationship of constitutive and inducible liver GST expression level in some of the tested fish species to microcystin exposure, a molecular mechanism for different microcystin detoxification abilities of the warm freshwater fishes was discussed.     

GST profile expression study in some selected plants: in silico approach

Glutathione acts as a protein disulphide reductant, which detoxifies herbicides by conjugation, either spontaneously or by the activity of one of a number of glutathione-S-transferases (GSTs), and regulates gene expression in response to environmental stress and pathogen attack. GSTs play roles in both normal cellular metabolisms as well as in the detoxification of a wide variety of xenobiotic compounds, and they have been intensively studied with regard to herbicide detoxification in plants. A newly discovered plant GST subclass has been implicated in numerous stress responses, including those arising from pathogen attack, oxidative stress and heavy-metal toxicity. In addition, plants GSTs play a role in the cellular response to auxins and during the normal metabolism of plant secondary products like anthocyanins and cinnamic acid. The present work involves two in silico analytical approaches—general secondary structure prediction studies of the proteins and detailed signature pattern studies of some selected GST classes in Arabdiopsis thaliana, mustard, maize and bread wheat by standard Bioinformatics tools; structure prediction tools; signature pattern tools; and the evolutionary trends were analyzed by ClustalW. For this purpose, sequences were obtained from standard databases. The work reveals that these proteins are mainly alpha helical in nature with specific signature pattern similar to phosphokinase C, tyrosine kinase and casein kinase II proteins, which are closely related to plant oxidative stress. This study aims to comprehend the relationship of GST gene family and plant oxidative stress with respect to certain specific conserved motifs, which may help in future studies for screening of biomodulators involved in plant stress metabolism.     

GST profile expression study in some selected plants: in silico approach

Glutathione acts as a protein disulfide reductant, which detoxifies herbicides by conjugation, either spontaneously or by the activity of one of a number of glutathione-S-transferases (GSTs), and regulates gene expression in response to environmental stress and pathogen attack. GSTs play role in both normal cellular metabolism as well as in the detoxification of a wide variety of xenobiotic compounds, and they have been intensively studied with regard to herbicide detoxification in plants. A newly discovered plant GST subclass has been implicated in numerous stress responses, including those arising from pathogen attack, oxidative stress, and heavy-metal toxicity. In addition, plant GSTs play a role in the cellular response to auxins and during the normal metabolism of plant secondary products like anthocyanins and cinnamic acid. The present study involves two in silico analytical approaches—general secondary structure prediction studies of the proteins and detailed signature pattern studies of some selected GST classes in Arabdiopsis thaliana, Mustard, Maize, and Bread wheat by standard Bioinformatics tools; structure prediction tools; signature pattern tools; and the evolutionary trends were analyzed by ClustalW. For this purpose, sequences were obtained from standard databases. The study reveals that these proteins are mainly alpha helical in nature with specific signature pattern similar to phosphokinase C, tyrosine kinase, and casein kinase II proteins, which are closely related to plant oxidative stress. This study aims to comprehend the relationship of GST gene family and plant oxidative stress with respect to certain specific conserved motifs, which may help in future studies for screening of biomodulators involved in plant stress metabolism.     

Characterization and molecular cloning of a glutathione S-transferase gene from the tick, Boophilus microplus (Acari: Ixodidae).

A glutathione S-transferase (GST) was purified from the larval cattle tick, Boophilus microplus (Acari: Ixodidae), by glutathione-affinity chromatography. The purified enzyme appeared as a single band on SDS-PAGE and has a molecular mass of 25.8 kDa determined by mass spectrometry. The N-terminus of the purified enzyme was sequenced. The full-length cDNA of the enzyme was isolated by RT-PCR using degenerate oligonucleotides derived from the N-terminal amino acid sequence. The cDNA contains an open reading frame encoding a 223-amino-acid protein with the N-terminus identical to the purified GST. Comparison of the deduced amino acid sequence with GSTs from other species revealed that the enzyme is closely related to the mammalian mu class GST.     

甲氧虫酰肼对不同抗性棉铃虫种群谷胱甘肽S-转移酶活性和基因表达量的影响

【目的】明确与谷胱甘肽S-转移酶（GST）相关联的棉铃虫 Helicoverpa armigera (Hübner)对甲氧虫酰肼的抗性分子机制, 更有效地开展棉铃虫抗药性的快速监测。【方法】用LC₄₀甲氧虫酰肼处理棉铃虫3龄初幼虫, 测定处理前、后抗性种群GST的活性及5种GST基因的表达量变化, 并比较一种Delta基因GSTd1的编码区序列。【结果】经测序、比对, 抗甲氧虫酰肼种群（R-methoxyfenozide）和同源对照种群（S-methoxyfenozide）GSTd1基因编码区序列相同, 表明其编码的酶结构没有发生改变。甲氧虫酰肼处理前, R-methoxyfenozide种群的GST比活力显著高于S-methoxyfenozide种群; 而药剂处理后, 两种群的GST比活力均降低, 但R-methoxyfenozide种群的活性可以快速回升。药剂处理前, R-methoxyfenozide种群的GST基因表达量显著高于S-methoxyfenozide种群。药剂处理对不同抗性种群GST基因表达量的影响差异较大。除了GSTs1, S-methoxyfenozide种群GST基因表达量均降低, 其中GSTd2和GSTe2可以缓慢回升。GSTs1表达量在36 h内没有明显变化, 但在48 h显著升高。R-methoxyfenozide种群的GST基因表达量均先降低, 然后迅速恢复, 除GSTe2基因外, R-methoxyfenozide种群的基因初始表达量和药剂处理后的最终表达量均显著高于S-methoxyfenozide种群。【结论】棉铃虫对甲氧虫酰肼的抗性与GST比活力增强有关, 而GST比活力的增强主要源于多个GST基因的过量表达。     

Isolation and Characterization of a cDNA Clone Coding for a Glutathione S-Transferase Class Delta Enzyme from the Biting Midge Culicoides variipennis sonorensis Wirth and Jones

Culicoides variipennis sonorensis is the primary vector of bluetongue viruses in North America. Glutathione S-transferases (GSTs) are enzymes that catalyze nucleophilic substitutions, converting reactive lipophilic molecules into soluble conjugates. Increased GST activity is associated with development of insecticide resistance. Described here is the isolation of the first cDNA encoding a C. variipennis GST. The clone consists of 720 translated bases encoding a protein with a M(r) of approximately 24,800 composed of 219 amino acids. The deduced amino acid sequence is similar (64%-74%) to class Delta (previously named Theta) GSTs from the dipteran genera Musca, Drosophila, Lucilia and Anopheles. The cDNA was subcloned into pET-11b, expressed in Epicurian coli BL21 (DE3) and has a specific activity of approximately 28,000 units/mg for the substrate 1-chloro-2,4-dinitrobenzene.     

The gene for the microsomal glutathione S-transferase is on human chromosome 12

The microsomal glutathione S-transferase (GST) is a unique membrane-bound GST structurally distinct from the cytosolic GSTs. A cDNA encoding this 154 amino acid protein has recently been isolated and characterized. Using the cDNA as the hybridization probe, we now report the assignment of the human microsomal GST gene to chromosome 12 through the use of a panel of mouse-human somatic cell hybrid lines. This locus has recently been designated as GST 12. In addition, genomic Southern blotting data suggest that the human microsomal GST is encoded by a single- or very-low-copy gene. Therefore, the human GST gene superfamily resides on at least four separate chromosomes: 1 (GST 1), 6 (GST 2), 11 (GST 3), and 12 (GST 12).     

Purification,regulation and cloning of a glutathione transferase (GST) from maize resembling the auxin-inducible type-III GSTs

The glutathione transferases (GSTs) from maize (Zea mays L.) with activities toward the chloroacetanilide herbicide metolachlor and the diphenyl ether herbicide fluorodifen were fractionated into two pools based on binding to affinity columns. Pool 1 GSTs were retained on Orange A agarose and were identified as isoenzymes Zea mays (Zm) GST I-I, Zm GST I-II and Zm GST I-III, which have been described previously. Pool 2 GSTs selectively bound to S-hexyl-glutathione-Sepharose and were distinct from the pool 1 GSTs, being composed of a homodimer of 28.5 kDa subunits, termed Zm GST V-V, and a heterodimer of the 28.5 kDa polypeptide and a 27.5 kDa subunit, termed Zm GST V-VI. Using an antibody raised to Zm GST V-VI, a cDNA expression library was screened and a Zm GST V clone identified showing sequence similarity to the type-III auxin-inducible GSTs previously identified in tobacco and other dicotyledenous species. Recombinant Zm GST V-V showed high GST activity towards the diphenyl ether herbicide fluorodifen, detoxified toxic alkenal derivatives and reduced organic hydroperoxides. Antibodies raised to Zm GST I-II and Zm GST V-VI were used to monitor the expression of GST subunits in maize seedlings. Over a 24 h period the Zm GST I subunit was unresponsive to chemical treatment, while expression of Zm GST II was enhanced by auxins, herbicides, the herbicide safener dichlormid and glutathione. The Zm GST V subunit was more selective in its induction, only accumulating significantly in response to dichlormid treatment. During development Zm GST I and Zm GST V were expressed more in roots than in shoots, with Zm GST II expression limited to the roots.