Systematic analysis and functional verification of citrus glutathione S-transferases reveals that CsGSTF1 and CsGSTU18contribute negatively to citrus bacterial canker

Citrus bacterial canker(CBC) is resulted from Xanthomonas citri subsp. citri(Xcc) infection and poses a significant threat to citrus production.Glutathione S-transferases(GSTs) are critical in maintaining redox homeostasis in plants, especially in relation to abiotic and biotic stress responses. However, the function of GSTs in resisting CBC remains unclear. Here, citrus glutathione S-transferases were investigated applying a genome-wide approach. In total, 69 CsGSTs belonging to seven classes were identified, and the phylogeny, chromosomal distribution, gene structures and conserved motifs were analyzed. Several CsGSTs responded to Xcc infection, as observed in the upregulation of CsGSTF1 and CsGSTU18 in the CBC-sensitive ‘Wanjincheng' variety but not in the resistant ‘Kumquat' variety. CsGSTF1 and CsGSTU18 were localized at the cytoplasm. Transient overexpression of CsGSTF1 and CsGSTU18 mediated reactive oxygen species(ROS) scavenging, whereas the virus-induced gene silencing(VIGS) of CsGSTF1 and CsGSTU18 caused strong CBC resistance and ROS burst. The present study investigated the characterization of citrus GST gene family, and discovered that CsGSTF1 and CsGSTU18 negatively contributed to CBC through modulating ROS homeostasis. These findings emphasize the significance of GSTs in infection resistance in plants.

Glutathione S-Transferase(GST)Identified from Giant Kelp Macrocystis pyrifera Increases the Copper Tolerance of Synechococcus elongatus PCC 7942

The glutathione S-transferases gene family plays an important regulatory role in growth and development,and responses to environmental change.In this study,six complete GST genes(Mp GST1,Mp GST2,Mp GST3,MpGST4,Mp GST5,and Mp GST6)were cloned from the gametophytes of brown alga Macrocystis pyrifera.Subsequent bioinformatics analysis showed that these six genes encoded proteins with 202,216,288,201,205,and 201 aa,respectively.Moreover,Mp GST3 differs from the other GST genes.Phylogenetic analysis suggested that MpGST3 belongs to the Ure2p type GST.Domain analysis suggested that the other GSTs from M.pyrifera belong to the soluble GST family and form an independent branch with the GSTs found in the other macroalgae,suggesting that a new GST type was formed during macroalgal evolution.GST genes were upregulated in M.pyrifera when 2.5 mg L^(-1)Cu ions were added to the medium.Six GST genes were integrated into the genome of Synechococcus elongatus PCC 7942,and their functions were verified by measuring light absorbance,photosynthetic pigment content,and photosynthetic parameters of the transformed strains under 0.3 mg L^(-1)Cu ion stress.The results showed much higher levels of various parameters in the transformed strains than in the wild strain.The transformed strains(with the MpGST genes)showed significantly enhanced resistance to Cu ion stress,while the wild strain almost died.The results of this study lay a theoretical foundation for further research on the Cu ion stress resistance function of GSTs in M.pyrifera.

Glutathione S-transferase(GST) gene expression profiles in two marine bivalves exposed to BDE-47 and their potential molecular mechanisms

Glutathione S-transferases （GSTs） are phase II enzymes that facilitate the detoxification of xenobioties and play important roles in antioxidant defense. We investigated the expression patterns of seven Venerupis philippinarum GSTs （VpGSTs） and four Mytilus galloprovincialis GSTs （MgGSTs） following exposure to BDE-47. Differential expressions of the seven VpGSTs and four MgGSTs transcripts were observed, with differences between the hepatopancreas and gills. Among these GSTs, the sigma classes （VpGSTS1, VpGSTS2, VpGSTS3, MgGST1, and MgGST3） were highly expressed in response to BDE-47 exposure, demonstrating their potential as molecular biomarkers for environmental biomonitoring studies. We obtained the three-dimensional crystal structures of VpGSTs and MgGSTs by homologous modeling. A model to elucidate the binding interactions between the ligands and receptors was defined by molecular docking, Hydrophobic and n were the most often observed interactions between BDE-47 and the GSTs.

Anticancer Drug Resistance of HeLa Cells Transfected With Rat Glutathione S-transferase pi Gene

To establish a cytologic expressing system of rat glutathione S-transferase pi (GST-pi) cDNA for detecting the resistance of HeLa cells to anticancer drugs. Methods The assessment was made with various anticancer drugs (adriamycin, mitomycin, cisplatinum and vincristine) that showed different cytotoxicities in transfectant HeLa cells with pSV-GT containing rat GST-pi cDNA (HeLa/pSV-GT) or control pSV-neo (HeLa/pSV-neo). Expression levels of GST-pi mRNA in HeLa/pSV-GT and HeLa/pSV-neo were measured by in situ hybridization using Digoxin-labelled cDNA probe. Results HeLa/pSV-GT expressed significantly high degree of GST-pi mRNA, whereas both HeLa/pSV-neo and HeLa cells had very low expression. Cytotoxicities of HeLa/pSV-GT and HeLa/pSV-neo with 4 anticancer drugs were measured by MTT assay. Drug concentrations for yielding 50% inhibition (IC50) in HeLa/pSV-GT by adriamycin, mitomycin and cisplatinum were 70.13 靏/mL, 10.95 靏/mL and 16.52 靏/mL, respectively. In contrast, IC50 in HeLa/pSV-neo was 10.34 靏/mL, 7.48 靏/mL and 13.70 靏/mL, respectively. The cytotoxicities of vincristine on both HeLa/pSV-GT and HeLa/pSV-neo were not significantly different. Conclusions Our findings suggest that HeLa/pSV-GT containing rat GST-pi cDNA is resistant to some anticancer drugs due to overexpression of GST-pi. Also, HeLa/pSV-GT cell line could serve as a useful cytogenetic model for further research.

Cloning, characterization and expression analysis of a microsomal glutathione S-transferase gene from the seagrass Zostera marina

The response of glutathione S-transferase(GST)in Zostera marina to temperature variation was analyzed at molecular level by cloning the microsomal GST gene and texting the microsomal GST expression regularity under different temperature.Specific speaking,express ZmGST in Escherichia coli,then purify the recombinant protein and make the thermal stability analysis.Therefore,the experiments were carried out to provide a theoretical basis for the further elaboration to the population degradation mechanisms of Z.marina.In conclusion,the thermostability and the response of ZmGST gene to temperature changes can determine its temperature tolerance range,and affect its resilience in turn.

Association of glutathione S-transferase T1 and M1 gene polymorphisms with ischemic stroke risk in the Chinese Han population

Atherosclerosis plays an important role in ischemic stroke, and oxidative stress participates in the entire process of atherosclerosis. Glutathione S-transferase （GST） acting with other antioxidant enzymes can eliminate reactive oxygen species and protect cells against oxidative damage. To assess the association of glutathione S-transferase （GSTT1 and GSTM1） gene polymorphisms with ischemic stroke in the Chinese Han population, the present study selected 315 patients with ischemic stroke and 210 healthy controls for comparison. GSTT1 and GSTM1 genotypes were determined using polymerase chain reactions, electrophoresis and imaging analysis. No obvious evidence of GSTTI-nulI, GSTMI-null and GSTTI/GSTMI-double null genotype distribution differences was found between case and control groups or between genders. Subgroup analysis showed that the risk of stroke was increased when hypertension was accompanied by GSTTl-null （odds ratio （OR） = 2.996, P 〈 0.001） and GSTMl-null （OR = 3.680, P 〈 0.001 ） genotypes; diabetes mellitus was accompanied by GSTTI-null （OR = 1.860, P = 0.031） and GSTMI-null （OR = 2.444, P = 0.002） genotypes, and smokers showed a GSTTl-null genotype （OR = 2.276, P = 0.003）. GSTT1- and GSTMl-null genotypes may interact synergistically with hypertension, diabetes mellitus and smoking to increase the incidence risk of ischemic stroke.

Genetic Polymorphisms Analysis of Glutathione S-transferase M1 and T1 in Children with Acute Lymphoblastic Leukemia

Summary: The relationship between glutathione S-transferases (GSTs) M1, T1 genotype and childhood acute lymphoblastic leukemia (ALL) was investigated. GSTM1 and GSTT1 genotypes in genomic DNA from 67 children with ALL and 146 healthy controls were analyzed by using the multiplex polymerase chain reaction (PCR). The frequencies of GSTM1, M1-T1 null genotypes in ALL children were significantly higher than in the healthy controls (76.12 % versus 52.74 %, OR=2.856, P<0.001; 50.74 % versus 24.66 %, OR=3.148, P<0.001, respectively). However, there was no significant relationship between GSTT1 null genotype and ALL of children (61.19 % versus 49.32 %, OR=1.621, P>0.05). It was suggested that GSTM1 null genotype might be a risk genotype of childhood ALL, while there as no correlation between GSTT1 null genotype and childhood ALL.

Cloning and Characterization of a Tau Glutathione S-transferase Subunit Encoding Gene in Gossypium hirsutum

A predicted tau glutathione S-transferase(GST) subunit encoding gene,named GhGST,was isolated from Gossypium hirsutum with RACE method from SSH library based on Verticillium

Association among Serum Organochlorine Pesticide Residues, Glutathione S-Transferase M1 Genetic Polymorphism and Female Breast Cancer

Background: The purpose of this study was to evaluate the association among serum organochlorine pesticide residues, glutathione S-transferase M1 genetic polymorphism and female breast cancer. Methods: A 1:1 matched case-control study of 140 newly diagnosed breast cancer patients and 140 non-cancer female patients who consulted the five largest hospitals in the Tangshan city from September 2006 to October 2007. Results: The result showed higher risk of breast cancer among subjects with higher levels of serum DDT and HCH residue, the OR was 3.18 (95%CI, 1.11 - 9.07) and 5.02 (95%CI, 1.64 - 16.56).The value of ORe associated with single environmental factor DDT high residues, and ORg associated with single GSTM1 deletion genotype were respectively 3.86 (1.20 - 12.47) and 1.34 (0.36 - 5.08). The OReg associated with combined action of two factors was 5.59 (1.63 - 18.90), and the value of interaction parameters (γ) equaled 1.24. The value of ORe associated with single environmental factor HCH higher residue and ORg associated with single GSTM1 deletion genotype were respectively 2.73 (0.84 - 8.87) and 1.48 (0.49 - 4.60). The value of OReg associated with combined action of two factors was 3.87 (1.18 - 12.68), and γ equaled 1.38. Conclusion: The results indicated that breast cancer occurrence was the combined result of environmental and genetic factors. The concurrent action of GSTM1 deletion genotype and DDT/HCH enhanced the risk of breast cancer.

Polymorphisms of the oxidant enzymes glutathione S-transferase and glutathione reductase and their association with resistance of Plasmodium falciparum isolates to antimalarial drugs

Objective:To investigate the association between amplification of the two regulatory genes controlling glutathione(GSH) levels,glutathione reductase(PfGR) and glutathione S-transferase (PfGST) genes and sensitivity of Plasmodium falciparum(P.falciparum) isolates collected from different malaria endemic areas of Thailand to standard antimalarial drugs.Methods:A total of 70 P.falciparum isolates were collected from endemic areas of multi-drug resistance (Tak,Chantaburi and Ranong Provinces) during the year 2008-2009.The in vitro assessment of antimalarial activity of P.falciparum clones(K1- and Dd2 chloroquine resistant and 3D7- chloroquine sensitive) and isolates to chloroquine,quinine,mefloquine and arteusnate was performed based on SYBR Green modified assay.Results:68(97.14%),11(15.71%) and 28(40%) isolates respectively were classified as chloroquine-,quinine- and mefloquine-resistant isolates. With this limited number of P.falciparum isolates included in the analysis,no significant association between amplification of PfGST gene and sensitivity of the parasite to chloroquine, quinine,mefloquine and quinine was found.Based on PCR analysis,Dd2,Kl and 3D7 clones all contained only one copy of the PfGST gene.All isolates(70) also carried only one copy number of PfGST gene.There appears to be an association between amplification of PfGR gene and chloroquine resistance.The 3D7 and Dd2 clones were found to carry only one PfGR gene copy, whereas the K1 clone carried two gene copies.Conclusions:Chloroquine resistance is likely to be a consequence of multi-factors and enzymes in the GSH system may be partly involved. Larger number of parasite isolates are required to increase power of the hypothesis testing in order to confirm the involvement of both genes as well as other genes implicated in glutathione metabolism in conferring chloroquine resistance.

Comparative Studies of Substrate and Inhibitor Specificity of Glutathione S-Transferases in Six Tissues of Oxya chinensis(Thunberg)(Orthoptera:Acrididae)

Specific activity, substrate specificity, and kinetic parameters （Km and Vmax） of glutathione S-transferases （GSTs） towards three substrates, 1-chloro-2,4-dinitrobenzene （CDNB）, 1,2-dichloro-4-nitrobenzene （DCNB）, and p-nitrobenzene chloride （pNBC） were investigated in six tissues （foregut, midgut, hindgut, fat body, hemolymph, and muscle） of Oxya chinensis. In addition, the inhibition in vitro （ethacrynic acid, and Cibacron Blue 3GA） of Oxya chinensis in the six tissues was also investigated. Glutathione S-transferase activity was detected in all the six tissues examined. The rank order of GST activities towards CDNB was fat body 〉 midgut 〉 hindgut 〉 muscle 〉 foregut 〉 hemolymph both in females and males. Glutathione S-transferase activities in the fat body in females and males were 1.3- to 10.4-fold and 1.1- to 10.0- fold higher than those in the other tissues. The rank order of GST activities towards the other substrates changed slightly. From these results, it was inferred that GSTs in the fat body and midgut played important roles in detoxifying xenobiotics including insecticides and plant allelochemicals in O. chinensis. In the three substrates examined, CDNB seemed to be the best substrate, followed by pNBC and DCNB. The kinetic parameters of GSTs were different among the six tissues. This suggested that GSTs in different tissues have various affinities and catalytic efficiency to substrates. In vitro inhibition study showed that the median inhibition concentration （IC50） values of the two inhibitors to GSTs from the six tissues were different. The results suggested that the two inhibitors have different inhibition potency to GSTs from the different tissues. The observed changes in kinetic parameters and inhibition in vitro among the six tissues of the insect might suggest that the number and structure of isoenzymes and their rate of expression varied for the different tissues.

Expression and Characterization of a Sigma-Class Glutathione S-transferase of the Oriental Migratory Locust, Locusta migratoria manilensis (Meyen)

A cDNA encoding a sigma-class glutathione S-transferase of the locust, Locusta migratoria manilensis （LmGSTs1）, was cloned by reverse transcriptase-polymerase chain reaction. The 830 bp-long cDNA encoded a 615 bp open reading frame （204 amino acid polypeptide）, which exhibited the structural motif and domain organization characteristic of GST sigma-class. It revealed 59, 57, 57, and 56% identities to sigma-class GSTs from Blattella germanica, Gryllotalpa orientalis, Nasonia vitripennis, and Pediculus humanus corporis, respectively. A recombinant protein （LmGSTs1） was functionally expressed in Escherichia coli cells in a soluble form and purified to homogeneity. LmGSTs1 was able to catalyze the biotranslation of glutathione with 1-chloro-2,4-dinitrobenzene, a model substrate for GSTs, as well as with p-nitro-benzyl chloride. Its optimal activity was observed at pH 8.0 and at 30℃. Incubation for 30 min at temperatures below 50℃ scarcely affected the activity. The I50 of reactive blue （RB） was 18.5 μmol L-1. In the presence of 0.05 mmol L-1 ethacrynic acid （ECA）, LmGSTs1 showed （81±3）% of the original activities.

ENZYME-LINKED IMMUNOSORBENT ASSAY OF HUMAN PLACENTA TYPE GLUTATHIONE S-TRANSFERASE AND ITS APPLICATION IN THE DIAGNOSIS OF HEPATOCARCINOMA

GST-π was purified from human placenta and its antiserum was raised in rabbits. The antibody IgC was purified and degraded into Fab' fragment which was conjugated with horseradish peroxidase (HRP) using N-succinimidyl-4-(N-maleimido-methyl) cyclo-hexane-1-carboxylate (SMCC) as crosslinking reagent to produce Fab'-HRP conjugate. A sandwich ELISA was established for the microquantitative determination of GST-π. The sensitivity was 11 pg/tube, which was far more sensitive than the radioimmunoassay so far reported. Using this method, the serum GST-π of 41 cases normal adult was found to be 1.06±0.94 ng/ml. The upper limit of the normal value was 2.6 ng/ml. In 30 cases of primary hepatocarcinoma, the level of serum GST-π was 24.4± 17.4 ng/ml, which was 23 times higher than the normal average value (P<0.01). The positive rate was 90%. In contrast, serum GST-π in 25 cases of chronic hepatitis was determined to be 1.74±1.16 ng/ml, which was not significantly different from the normal value (P>0.05). The pseudo-positive rate was 12.0%.

Frequency of Null Phenotypes of Glutathione S-Transferase M1 and T1 among the Populations of Tabuk (Northwestern Part of Saudi Arabia)

Background: The variability in the distribution of the null phenotypes of GSTM1 and GSTT1, due to total or partial gene deletion resulting in the lack of the active enzyme, has been reported in different populations, especially in ethnically well-defined groups but not in Tabuk. This study investigated the variability in the distribution of the null phenotypes of GSTM1 and GSTT1 in the population of Tabuk (northwestern part of Saudi Arabia). Method: This study was conducted on 200 subjects of Tabuk—northwestern part of Saudi Arabia among which 100 were chronic smokers and 100 were nonsmokers. The subjects were reporting to hospital for routine checkup. All were without past history of any chronic disease and no significant abnormality. GST genotyping was done by multiplex PCR-based methods. The smoker and control groups were compared using a chi-square test with P GSTM1 deletion homozygosity of 14% and 1% was reported among non smokers and smokers, respectively whereas GSTT1 deletion homozygosity of 28% and 6% was reported among non smokers and smokers, respectively. Our results indicate that there are major differences in allelic distribution of GSTM1 and GSTT1 genes between the two groups investigated. Combined analysis of both genes revealed that 15% of smokers and non smokers harbor the deleted genotype of GSTM1 and 34% of smokers and non smokers harbor the deleted genotype of GSTT1 with significant differences. Conclusion: This study enables selecting subgroups among the general population who are more susceptible to DNA damage and will help genetic studies on the association of GST polymorphisms with disease risks and drug effects in Arab population. Studies with a larger sample size are needed to evaluate and confirm the validity of our results.

星豹蛛两个GST基因克隆、鉴定及其对溴氰菊酯胁迫的响应表达

【目的】为明确星豹蛛Pardosa astrigera体内2个谷胱甘肽S-转移酶(glutathione S-transferases,GSTs)基因的时空表达模式,并探究其是否能对溴氰菊酯的胁迫做出响应。【方法】基于星豹蛛转录组数据库,PCR克隆星豹蛛GST基因PaGSTd1和PaGSTd2全长cDNA序列并进行生物信息学分析;利用RT-qPCR检测PaGSTd1和PaGSTd2在星豹蛛不同发育阶段(2-6龄若蛛和成蛛)、雌雄成蛛不同组织(头胸部、腹和足)以及通过药膜法利用不同浓度[LC 10(5.151 mg/L),LC 30(8.619 mg/L)和LC 50(12.311 mg/L)]溴氰菊酯胁迫不同时间(6,12,18,24和48 h)雄成蛛中的表达量。【结果】星豹蛛PaGSTd1(GenBank登录号:OR096398)全长cDNA序列长708 bp,开放阅读框长645 bp,编码214个氨基酸;星豹蛛PaGSTd2(GenBank登录号:OR096399)全长cDNA序列长793 bp,开放阅读框长645 bp,编码214个氨基酸。RT-qPCR检测结果表明,PaGSTd1和PaGSTd2在星豹蛛不同发育阶段和成蛛不同组织都有表达,均在5龄若蛛中的表达量最低;PaGSTd1在4龄若蛛中的表达量最高,PaGSTd2在成蛛中的表达量最高;PaGSTd1和PaGSTd2在足中的表达量最高,在除腹部外的其他组织中表达量均为雄成蛛的显著高于雌成蛛的。LC 10溴氰菊酯胁迫24 h时星豹蛛雄成蛛中PaGSTd1被诱导表达,6和18 h时PaGSTd2被诱导表达;LC 30溴氰菊酯胁迫18,24和48 h时雄成蛛中PaGSTd1被诱导表达,18 h时PaGSTd2被诱导表达;LC 50溴氰菊酯胁迫24 h时雄成蛛中PaGSTd1和PaGSTd2被诱导表达。【结论】本研究克隆得到星豹蛛两个GST基因PaGSTd1和PaGSTd2,均在星豹蛛足中表达量最高,说明足中这2个GST基因在对外源物质的解毒起到重要作用;这两个GST基因可以被溴氰菊酯诱导表达,说明可能参与星豹蛛对溴氰菊酯的胁迫响应,为后续GSTs的功能研究提供线索。

Effect of trans-acting factor on rat glutathione S-transferase P1 gene transcription regulation in tumor cells

Objective To investigate the effect of trans-acting factor(s) on rat glutathione S-transferase P1 gene (rGSTP1) transcription regulation in tumor cells.Methods The binding of trans-acting factor(s) to two enhancers of the rGSTP1 gene, glutathione S-transferase P enhancer Ⅰ (GPEI) and glutathione S-transferase P enhancer Ⅱ-1 (GPEⅡ-1), was identified by an electrophoretic mobility shift assay (EMSA). The molecular weight of trans-acting factor was measured in a UV cross-linking experiment. Results Trans-acting factor interacting with the core sequence of GPEI (cGPEI) were found in human cervical adenocarcinoma cell line (HeLa) and rat hepatoma cell line (CBRH7919). These proteins were not expressed in normal rat liver. Although specific binding proteins that bound to GPEⅡ-1 were detected in all three cell types, a 64 kDa binding protein that exists in HeLa and CBRH7919 cells was absent in normal rat liver. Conclusion cGPEI, GPEII specific binding proteins expressed in HeLa and CBRH7919 cells may play an important role in the high transcriptional level of the rGSTP1 gene in tumor cells.

Glutathione S-transferase genes in scallops and their diverse expression patterns after exposure to PST-producing dinoflagellates

The glutathione S-transferases(GSTs)are a superfamily of enzymes that function in cellular protection against toxic substances and oxidative stress.Bivalves could accumulate high concentration of paralytic shellfish toxins(PSTs)from harmful algae.To understand the possible involvement of GSTs in protecting bivalves during PST accumulation and metabolism,the GST genes were systemically analyzed in two cultured scallops,Azumapecten farreri and Mizuhopecten yessoensis,which were reported for PST deposition during harmful algae bloom.A total of 35 and 37 GSTs were identified in A.farreri(AfGSTs)and M.yessoensis(MyGSTs)genome,respectively,and the expansion of the sigma class from the cytosolic subfamily was observed.In both scallop species,sigma class GSTs showed higher expression than other members.The high GSTs expression was detected mainly during/after larvae stages and in the two most toxic organs,hepatopancreas and kidney.After ingesting PST-producing dinoflagellates,all the regulated AfGSTs in the hepatopancrcas were from the sigma class,but with opposite regulation pattern between Alexandrium catenella and A.minutum exposure.In scallop kidneys,where PSTs transformed into higher toxicity,more AfGSTs were regulated than in the hepatopancreas,and most of them were from the sigma class,with similar regulation pattern between A.catenella and A.minutum exposure.In M.yessoensis exposed to A.catenella,MyGST-σ2 was the only up-regulated MyGST in both hepatopancreas and kidney.Our results suggested the possible diverse function of scallop GSTs and the importance of sigma class in the defense against PSTs,which would contribute to the adaptive evolution of scallops in marine environments.

木豆抗旱相关基因CcGST1克隆与表达分析

为探讨谷胱甘肽转移酶(GST)的抗逆机理,克隆木豆中的抗旱谷胱甘肽转移酶基因,基于木豆干旱胁迫c DNA文库中1个GST基因相关的EST片段,利用RACE技术从木豆幼苗中克隆了该基因,命名为CcGST1,研究其在不同组织及不同干旱时期的表达特性。结果表明:该基因含1个669 bp的开放阅读框,编码223个氨基酸残基,含有保守的谷胱甘肽-S-转移酶的N端和C端结构域;系统进化树分析结果表明木豆CcGST1与大豆、宽叶菜豆等豆科植物的GST亲缘关系较近;亚细胞定位预测CcGST1蛋白可能定位于叶绿体;荧光定量PCR分析表明,CcGST1在木豆幼苗根、茎和叶中均有表达,但根中的表达水平最高,同时该基因受干旱胁迫诱导表达。据此推测木豆CcGST1基因可能与木豆应激干旱胁迫相关。

肺结核标准治疗患者GSTs基因多态性与抗结核药物诱导肝损伤的相关性分析

目的研究肺结核患者的GSTs(rs1045642)基因多态性与结核标准治疗方案治疗后抗结核药物诱导肝损伤(AT-DILI)之间的相关性。方法采用前瞻性研究方法,纳入2019年1月至2020年12月在成都中医药大学附属医院、四川省人民医院诊断为肺结核的住院治疗患者94例为研究对象。应用聚合酶链反应(PCR)-芯片杂交技术测定GSTs基因GSTM1、GSTT1、GSTP1的基因型。依据是否发生肝损伤,分为试验组(肝损伤组)27例和对照组(未发生肝损伤组)67例,收集两组患者基本资料并进行统计学处理分析,GSTM1、GSTT1、GSTM1&GSTT1、GSTP1基因多态性与AT-DILI的相关性。结果GSTM1、GSTT1基因多态性在试验组和对照组之间差异无统计学意义(P>0.05)。GSTP1的AA型、AG型、GG型基因多态性在试验组和对照组间比较差异有统计学意义(P<0.05),AA型与AG+GG型在试验组和对照组间差异有统计学意义(P<0.05)。结论GSTP1(rs1695)基因多态性是AT-DILI的危险因素。

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648bp,编码215个氨基酸,分子量约为24.47kDa,理论等电点为5.49;TuGSTd2的开放阅读框为648bp,编码215个氨基酸,分子量约为24.57kDa,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。