Over-expression of glutathione S-transferase(GST)can promote Cisplatin resistance in hepatocellular carcinoma(HCC)treatment.Hence,inhibiting GST is an attractive strategy to improve Cisplatin sensitivity in HCC therap...Over-expression of glutathione S-transferase(GST)can promote Cisplatin resistance in hepatocellular carcinoma(HCC)treatment.Hence,inhibiting GST is an attractive strategy to improve Cisplatin sensitivity in HCC therapy.Although several synthesized GST inhibitors have been developed,the side effects and narrow spectrum for anticancer seriously limit their clinical application.Considering the abundance of natural compounds with anticancer activity,this study developed a rapid fluorescence technique to screen“green”natural GST inhibitors with high specificity.The fluorescence assay demonstrated that schisanlactone B(hereafter abbreviated as C1)isolated from Xue tong significantly down-regulated GST levels in Cisplatin-resistant HCC cells in vitro and in vivo.Importantly,C1 can selectively kill HCC cells from normal liver cells,effectively improving the therapeutic effect of Cisplatin on HCC mice by downregulating GST expression.Considering the high GST levels in HCC patients,this compound demonstrated the high potential for sensitizing HCC therapy in clinical practice by down-regulating GST levels.展开更多
Jujube witches’broom(JWB)caused by phytoplasma has a severely negative effect on multiple metabolisms in jujube.The GST gene family in plants participates in the regulation of a variety of biotic and abiotic stresses...Jujube witches’broom(JWB)caused by phytoplasma has a severely negative effect on multiple metabolisms in jujube.The GST gene family in plants participates in the regulation of a variety of biotic and abiotic stresses.This study aims to identify and reveal the changes in the jujube GST gene family in response to phytoplasma infection.Here,70 ZjGSTs were identified in the jujube genome and divided into 8 classes.Among them,the Tau-class,including 44 genes,was the largest.Phylogenetic analysis indicated that Tau-class genes were highly conserved among species,such as Arabidopsis,cotton,chickpea,and rice.Through chromosome location analysis,37.1%of genes were clustered,and 8 of 9 gene clusters were composed of Tau class members.Through RT-PCR,qRT-PCR and enzyme activity detection,the results showed that the expression of half(20/40)of the tested ZjGSTs was inhibited by phytoplasma infection in field and tissue culture conditions,and GST activity was also significantly reduced.In the resistant and susceptible varieties under phytoplasma infection,ZjGSTU49-ZjGSTU54 in the cluster IV showed opposite expression patterns,which may be due to functional divergence during evolution.Some upregulated genes(ZjGSTU45,ZjGSTU49,ZjGSTU59,and ZjGSTU70)might be involved in the process of jujube against JWB.The yeast two-hybrid results showed that all 6 Tauclass proteins tested could form homodimers or heterodimers.Overall,the comprehensive analysis of the jujube GST gene family revealed that ZjGSTs responded actively to phytoplasma infection.Furthermore,some screened genes(ZjGSTU24,ZjGSTU49-52,ZjGSTU70,and ZjDHAR10)will contribute to further functional studies of jujube-phytoplasma interactions.展开更多
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 homeos...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.展开更多
文摘【目的】含有VQ基序(VQ)的蛋白质是植物特异性蛋白质,具有保守的“FxxhVQxhTG”氨基酸序列,调节植物生长和发育。番茄SlMPK1在高温胁迫过程中发挥重要作用,酵母双杂交(Y2H)显示SlVQ6蛋白能够与SlMPK1相互作用,通过GST pull-down技术进一步验证SlMPK1与SlVQ6的互作以及SlVQ6的互作网络,为研究SlVQ6介导的高温应答信号通路奠定基础。【方法】通过pGEX-4T-1-PC-VQ6质粒构建,以含有目的基因SIVQ6的菌液为模板,设计特异性引物,扩增目的基因序列,将获得的目的基因VQ6克隆到载体pGEX-4T-1的BamHⅠ和NotⅠ的酶切位点之间,将获得的重组质粒pGEX-4T-1-PC-VQ6转入TOP10克隆菌株经IPTG(异丙基硫代半乳糖苷)诱导表达,通过GST柱亲和纯化获得目标蛋白PC-VQ6,进而获得预期GST-SlVQ6融合蛋白,通过体外磷酸化进一步确定SlVQ6是否是SlMPK1的下游底物;以GST-SlVQ6为诱饵蛋白固定于GST Sepharose Beads上,与番茄叶片总蛋白孵育后洗脱,收集洗脱液进行SDS-PAGE凝胶电泳验证,通过LC-MS/MS检测SlVQ6可能互作的候选蛋白,并对筛选的SIVQ6互作蛋白通过GO、KEGG和蛋白互作网络进行生物信息学分析。【结果】成功构建pGEX-4T-1-PC-SlVQ6基因重组表达质粒,并获得带有GST标签的GST-SlVQ6融合蛋白,其分子量大小约为54 kD;将GST-SlVQ6和His-SlMPK1进行体外磷酸化试验,SlMPK1能够磷酸化SlVQ6,而作为阴性对照的GST与SlMPK1无相互作用,且SlVQ6不存在自磷酸化的现象,表明GST-SlVQ6和His-SlMPK1具有相互作用,且SlVQ6是SlMPK1的下游底物;以GST-SlVQ6融合蛋白为诱饵蛋白(空GST为阴性对照),利用pull-down试验筛选番茄叶片组织蛋白中与SlVQ6蛋白结合的蛋白,经SDS-PAGE分离、液相色谱-串联质谱(LC-MS/MS)鉴定以及Mascot与蛋白数据库检索,共鉴定出37个与SlVQ6结合的蛋白,包括蛋白激酶Receptor for Activated C Kinase 1B(RACK1B),通过GO、KEGG和蛋白互作网络的生物信息学分析,表明这些蛋白参与多种信号通路,其中8个核糖体蛋白可能与高温胁迫密切相关。【结论】SlVQ6是SlMPK1的底物蛋白,且37个蛋白可能与SlVQ6互作,这些蛋白与胁迫反应有着密切的联系,可能会在提高番茄植株高温耐受性等方面发挥重要作用。
基金supported by the National Natural Science Foundation of China(Grant Nos.:82003931,82204766 and 81374062)the Outstanding Youth Foundation of Hunan Provincial Education Department of China(Grant No.:20B445)+3 种基金the Hunan Youth Science and Technology Innovation Talents Project,China(Grant No.:2021RC3100)the Chinese Postdoctoral Science foundation(Grant No.:2021M690974)Changjiang Scholars Program in Ministry Education,People's Republic of China(Program No.:T2019133)the Scientific Research Project of Hunan Provincial Education Department(Project No.:21B0394).
文摘Over-expression of glutathione S-transferase(GST)can promote Cisplatin resistance in hepatocellular carcinoma(HCC)treatment.Hence,inhibiting GST is an attractive strategy to improve Cisplatin sensitivity in HCC therapy.Although several synthesized GST inhibitors have been developed,the side effects and narrow spectrum for anticancer seriously limit their clinical application.Considering the abundance of natural compounds with anticancer activity,this study developed a rapid fluorescence technique to screen“green”natural GST inhibitors with high specificity.The fluorescence assay demonstrated that schisanlactone B(hereafter abbreviated as C1)isolated from Xue tong significantly down-regulated GST levels in Cisplatin-resistant HCC cells in vitro and in vivo.Importantly,C1 can selectively kill HCC cells from normal liver cells,effectively improving the therapeutic effect of Cisplatin on HCC mice by downregulating GST expression.Considering the high GST levels in HCC patients,this compound demonstrated the high potential for sensitizing HCC therapy in clinical practice by down-regulating GST levels.
基金supported by grants from the National Key R&D Program Project Funding(Grant No.2018YFD1000607)the Foundation for 100 Innovative Talents of Hebei Province(Grant No.SLRC2019031)+1 种基金the National Natural Science Foundation of China(Grant No.31772285)the Hebei Province Innovation Foundation for Postgraduates(Grant No.CXZZBS2020097)。
文摘Jujube witches’broom(JWB)caused by phytoplasma has a severely negative effect on multiple metabolisms in jujube.The GST gene family in plants participates in the regulation of a variety of biotic and abiotic stresses.This study aims to identify and reveal the changes in the jujube GST gene family in response to phytoplasma infection.Here,70 ZjGSTs were identified in the jujube genome and divided into 8 classes.Among them,the Tau-class,including 44 genes,was the largest.Phylogenetic analysis indicated that Tau-class genes were highly conserved among species,such as Arabidopsis,cotton,chickpea,and rice.Through chromosome location analysis,37.1%of genes were clustered,and 8 of 9 gene clusters were composed of Tau class members.Through RT-PCR,qRT-PCR and enzyme activity detection,the results showed that the expression of half(20/40)of the tested ZjGSTs was inhibited by phytoplasma infection in field and tissue culture conditions,and GST activity was also significantly reduced.In the resistant and susceptible varieties under phytoplasma infection,ZjGSTU49-ZjGSTU54 in the cluster IV showed opposite expression patterns,which may be due to functional divergence during evolution.Some upregulated genes(ZjGSTU45,ZjGSTU49,ZjGSTU59,and ZjGSTU70)might be involved in the process of jujube against JWB.The yeast two-hybrid results showed that all 6 Tauclass proteins tested could form homodimers or heterodimers.Overall,the comprehensive analysis of the jujube GST gene family revealed that ZjGSTs responded actively to phytoplasma infection.Furthermore,some screened genes(ZjGSTU24,ZjGSTU49-52,ZjGSTU70,and ZjDHAR10)will contribute to further functional studies of jujube-phytoplasma interactions.
基金funded by the National Key Research and Development Program of China (Grant No.2022YFD1201600)Natural Science Foundation of Chongqing (Grant No.cstc2020jcyj-msxmX1064)+1 种基金Earmarked Funds for the China Agriculture Research System (Grant No.CARS-26)Three-year Action Plan of Xi'an University (Grant No.2021XDJH41)。
文摘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.
