Acetolactate synthase(ALS)-targeting herbicides are among the most widely used weed-control chemicals globally.Mutations in the ALS gene can confer herbicide resistance in crops,thereby allowing selective elimination ...Acetolactate synthase(ALS)-targeting herbicides are among the most widely used weed-control chemicals globally.Mutations in the ALS gene can confer herbicide resistance in crops,thereby allowing selective elimination of weeds without harming crops.Herbicide-resistant ALS alleles were initially discovered in weeds and subsequently developed through artificial mutagenesis techniques.With the advancement of CRISPR/Cas technologies,various genome-editing tools are now available to introduce these resistant alleles,as well as novel variants,into diverse crop species.Moreover,emerging methodologies,such as directed evolution,enable the generation and screening of large populations of random ALS mutants.Consequently,ALS has become one of the most extensively targeted genes in plant gene evolution.This paper provides a comprehensive overview of both conventional and recently developed strategies for ALS evolution,with particular emphasis on CRISPR/Cas-based genome editing and directed evolution.Future perspectives on technological application are also discussed.By advancing our understanding of herbicide-resistant ALS allele development for crop improvement,these methodologies may also pave the way for their application to the evolution of other agronomically important genes.展开更多
Carotenoids are natural pigments that are widely distributed in the flowers,fruits,and seeds of many plant species.These compounds not only endow diverse colors but also exhibit antioxidant,immune-modulatory,anti-agin...Carotenoids are natural pigments that are widely distributed in the flowers,fruits,and seeds of many plant species.These compounds not only endow diverse colors but also exhibit antioxidant,immune-modulatory,anti-aging,and photoprotective properties.Although carotenoid metabolism has been studied extensively in microbial and plant science,the genetic mechanisms underlying carotenoid metabolism in cotton remain underexplored.Here,we isolated gene GbDYA that regulates a dark-yellow anther color by map-based cloning using a BC1F1 population derived from a cross of Gossypium barbadense acc.Hai7124 with dark-yellow anthers and G.hirsutum acc.TM-1 with light-yellow anthers backcrossed with TM-1.GbDYA encodes phytoene synthase,a key rate-limiting enzyme in the carotenoid biosynthesis pathway.A long terminal repeat retrotransposon in the first exon of GhDYA(an ortholog of GbDYA in G.hirsutum acc.TM-1)caused loss of function and led to the light-yellow anther color.GbDYA is predominantly expressed in the early stages of anther development.Transcriptome,RT-qPCR and KEGG enrichment analyses revealed that GbDYA influences the synthesis and accumulation of carotenoids in anthers by modulating expression of key genes in the carotenoid biosynthesis pathway.Integrated transcriptomic and metabolomic analyses indicated that the accumulation of lutein,violaxanthin,antherxanthin,cryptoxanthin,zeaxanthin,andβ-carotene contributed to yellow coloration of anthers.Dual-luciferase and yeast one-hybrid assays confirmed that transcription factor GbMYB105(GB_A11G3511)binds to the promoter of GbDYA and activates its expression.High-temperature stress treatment indicated that carotenoids accumulation in anthers enhances pollen antioxidant activity.This study unravels the role of GbDYA in conferring the anther coloration,and provides the potential utilization by modulating accumulation of carotenoids in anthers to enhance pollen viability in high-temperature tolerance breeding in cotton.展开更多
Microglia,the resident monocyte of the central nervous system,play a crucial role in the response to spinal cord injury.However,the precise mechanism remains unclear.To investigate the molecular mechanisms by which mi...Microglia,the resident monocyte of the central nervous system,play a crucial role in the response to spinal cord injury.However,the precise mechanism remains unclear.To investigate the molecular mechanisms by which microglia regulate the neuroinflammatory response to spinal cord injury,we performed single-cell RNA sequencing dataset analysis,focusing on changes in microglial subpopulations.We found that the MG1 subpopulation emerged in the acute/subacute phase of spinal cord injury and expressed genes related to cell pyroptosis,sphingomyelin metabolism,and neuroinflammation at high levels.Subsequently,we established a mouse model of contusive injury and performed intrathecal injection of siRNA and molecular inhibitors to validate the role of ceramide synthase 5 in the neuroinflammatory responses and pyroptosis after spinal cord injury.Finally,we established a PC12-BV2 cell co-culture system and found that ceramide synthase 5 and pyroptosis-associated proteins were highly expressed to induce the apoptosis of neuron cells.Inhibiting ceramide synthase 5 expression in a mouse model of spinal cord injury effectively reduced pyroptosis.Furthermore,ceramide synthase 5-induced pyroptosis was dependent on activation of the NLRP3 signaling pathway.Inhibiting ceramide synthase 5 expression in microglia in vivo reduced neuronal apoptosis and promoted recovery of neurological function.Pla2g7 formed a“bridge”between sphingolipid metabolism and ceramide synthase 5-mediated cell death by inhibiting the NLRP3 signaling pathway.Collectively,these findings suggest that inhibiting ceramide synthase 5 expression in microglia after spinal cord injury effectively suppressed microglial pyroptosis mediated by NLRP3,thereby exerting neuroprotective effects.展开更多
Background:Under hypoxia,exaggerated compensatory responses may lead to acute mountain sickness.The excessive vasodilatory effect of nitric oxide(NO)can lower the hypoxic pulmonary vasoconstriction(HPV)and peripheral ...Background:Under hypoxia,exaggerated compensatory responses may lead to acute mountain sickness.The excessive vasodilatory effect of nitric oxide(NO)can lower the hypoxic pulmonary vasoconstriction(HPV)and peripheral blood pressure.While NO is catalyzed by various nitric oxide synthase(NOS)isoforms,the regulatory roles of these types in the hemodynamics of pulmonary and systemic circulation in living hypoxic animals remain unclear.Therefore,this study aims to investigate the regu-latory effects of different NOS isoforms on pulmonary and systemic circulation in hypoxic rats by employing selective NOS inhibitors and continuously monitoring hemodynamic parameters of both pulmonary and systemic circulation.Methods:Forty healthy male Sprague–Dawley(SD)rats were randomly divided into four groups:Control group(NG-nitro-D-arginine methyl ester,D-NAME),L-NAME group(non-selective NOS inhibitor,NG-nitro-L-arginine methyl ester),AG group(in-ducible NOS inhibitor group,aminoguanidine),and 7-NI group(neurological NOS in-hibitor,7-nitroindazole).Hemodynamic parameters of rats were monitored for 10 min after inhibitor administration and 5 min after induction of hypoxia[15%O2,2200 m a.sl.,582 mmHg(76.5 kPa),Xining,China]using the real-time dynamic monitoring model for pulmonary and systemic circulation hemodynamics in vivo.Serum NO concentra-tions and blood gas analysis were measured.Results:Under normoxia,mean arterial pressure and total peripheral vascular resist-ance were increased,and ascending aortic blood flow and serum NO concentration were decreased in the L-NAME and AG groups.During hypoxia,pulmonary arterial pressure and pulmonary vascular resistance were significantly increased in the L-NAME and AG groups.Conclusions:This compensatory mechanism activated by inducible NOS and en-dothelial NOS effectively counteracts the pulmonary hemodynamic changes induced by hypoxic stress.It plays a crucial role in alleviating hypoxia-induced pulmonary arte-rial hypertension.展开更多
Background:Lung cancer is one of the deadliest cancers worldwide,creating a pressing need to develop novel drugs that inhibit oncogenic signaling pathways.Numerous studies have shown that berberine(BBR)has anti–lung ...Background:Lung cancer is one of the deadliest cancers worldwide,creating a pressing need to develop novel drugs that inhibit oncogenic signaling pathways.Numerous studies have shown that berberine(BBR)has anti–lung cancer potential.We aimed to explore the anti–lung cancer effect of BBR and related mechanisms by targeting the glycogen synthase kinase 3β(GSK3β)/β-catenin pathway.Methods:Lung adenocarcinoma(LUAD)cells A549 and NCI-H1975 were treated with BBR.Results:Our results showed that BBR inhibited cell proliferation by decreasing c-Myc levels and induced cel cycle arrest in the G0/G1 phase by lowering cyclin D1 levels.BBR induced apoptosis by upregulating cleaved caspase 3 levels.BBR inhibited cell migration and invasion by decreasing N-cadherin levels.Furthermore,BBR upregulated the expression of GSK3βprotein and phospho-β-catenin proteins in the cytoplasm,while decreasing the expression ofβ-catenin protein.Next,LUAD cel s were exposed to CHIR-99021(a GSK3βinhibitor).This treatment led to an increase in c-Myc,cyclin D1,andβ-catenin levels at specific concentrations.BBR partially reversed the effects of CHIR-99021.Finally,LUAD cells were treated with CHIR-99021(4μmoL/L)combined with BBR(30 and 60μmoL/L)for 24 h.The expression of programmed death ligand 1(PD-L1)was assessed by Western blot analysis.Jurkat T cells and A549 cel s were cocultured for 24 h to examine the lactate dehydrogenase release rate.Results suggested that BBR suppressed the expression of PD-L1 and heightened the immune lethality of T cells.Conclusions:BBR suppressed the proliferative activity of LUAD cell lines A549 and NCI-H1975 in vitro,induced cell cycle arrest and cancer cel apoptosis in the G0/G1 stage,and repressed the migration and invasion of cancer cells.BBR reduced the PD-L1 protein expression and enhanced T-cell–mediated cytotoxicity.These effects appear to be related to BBR's regulation of the GSK3β/β-catenin pathway.展开更多
[Objective]To confirm the function of the farnesyl diphosphate(FPP)cyclase encoded by orf2064 in Streptomyces exfoliatus UC5319.[Methods]orf2064 was expressed in Escherichia coli,and the recombinant protein was purifi...[Objective]To confirm the function of the farnesyl diphosphate(FPP)cyclase encoded by orf2064 in Streptomyces exfoliatus UC5319.[Methods]orf2064 was expressed in Escherichia coli,and the recombinant protein was purified and assayed with FPP as the substrate.The reaction products were detected by GC-MS.An FPP-overproducing E.coli strain was engineered for heterologous expression of orf2064.The fermentation products were analyzed by GC-MS,and the target compound was isolated and structurally characterized by nuclear magnetic resonance spectroscopy(NMR).In addition,orf2064 was heterologously expressed in Streptomyces,and the fermentation products were analyzed by GC-MS.[Results]GC-MS revealed that both the in vitro reaction of the recombinant protein ORF2064 and the heterologous expression products in E.coli and Streptomyces consistently produced a compound with identical retention time and[M+]of m/z 204.Subsequent isolation,purification,and NMR analysis confirmed this compound as calarene.[Conclusion]The FPP cyclase encoded by orf2064 in S.exfoliatus is identified as an calarene synthase.展开更多
Isoflavones which mainly distributed in leguminous plants have plenty of health benefits.Isoflavone synthase(IFS)is a membrane-associated cytochrome P450 enzyme(CYP450)which carries out the unique aryl-ring migration ...Isoflavones which mainly distributed in leguminous plants have plenty of health benefits.Isoflavone synthase(IFS)is a membrane-associated cytochrome P450 enzyme(CYP450)which carries out the unique aryl-ring migration and hydroxylation.So far,few crystal structures of plant P450s have been obtained.We determined the crystal structure of IFS from Medicago truncatula at 1.9 by MAD method using a selenomethionine substituted crystal and conducted molecular docking and mutagenesis study.The structure of IFS complexed with imidazole exhibits the helix Iα-loop-helix Iβmotif which corresponds to helix I of other P 450s.Compared with structures of common P450s,IFS/imidazole structure contains an extra domain,i.e.,theγ-domain.The structure reveals a homodimer in which theγ-domain of one molecule interacts with theβ-domain of another.The plane of heme group makes an angle of approximately 40°with the helix Iα-loop-helix Iβmotif.Molecular docking combined with mutagenesis study suggested that Trp-128 and Asp-300 might play important roles in substrate binding and recognition.Phe-301,Ser-303 and Gly-305 from the helix Iα-loop-helix Iβmotif may play important roles in the aryl-ring migration.These novel structural features reveal insights into the unique reaction mechanism of IFS and provide a basis for engineering IFS in leguminous crops for health purpose.展开更多
Chitin is an abundant natural biopolymer that plays a crucial role in insect growth and development as a fundamental structural component of the exoskeleton.The membrane-integralβ-glycosyltransferase,chitin synthase,...Chitin is an abundant natural biopolymer that plays a crucial role in insect growth and development as a fundamental structural component of the exoskeleton.The membrane-integralβ-glycosyltransferase,chitin synthase,has been identified as the central component in chitin biosynthesis.However,the precise roles of other proteins in facilitating chitin synthase in chitin biosynthesis remain unclear.In this study,we employed split-ubiquitin membrane yeast two-hybrid(MYTH)and pull-down assays to demonstrate the physical interaction between Twinstar(Tsr),a small molecular protein in the actin-depolymerizing factor ADF/Cofilin protein family,and chitin synthase Krotzkopf verkehrt(Kkv)in Drosophila melanogaster in vitro.The RNA interference(RNAi)-mediated global knockdown of Tsr in D.melanogaster resulted in larval lethality.Furthermore,targeted suppression of Tsr in the tracheal and epidermal tissues also led to larval mortality,while knocking down Tsr in the wing tissues led to wrinkled wings.Additionally,silencing Tsr not only reduced the chitin content in the first longitudinal vein of the wings but also led to the absence of the chitin lamellar structure.To validate the functional conservation of Tsr in other insect orders,the two agricultural pests Ostrinia furnacalis and Tribolium castaneum,representing lepidoptera and coleoptera insects,respectively,were investigated.Knockdown experiments targeting the Drosophila Tsr orthologues OfTsr in O.furnacalis and TcTsr in T.castaneum produced abnormal larvae during molting or pupation in O.furnacalis and lethality in T.castaneum.Our findings not only improve ourknowledge of the chitin biosynthesis machinery in insect cuticles but also provide new potential targets for the control of major agricultural pests.展开更多
BACKGROUND Esophageal cancer(ESCA)is among the most prevalent and lethal tumors globally.While nitric oxide synthase 1(NOS1)is recognized for its important in-volvement in various cancers,its specific function in ESCA...BACKGROUND Esophageal cancer(ESCA)is among the most prevalent and lethal tumors globally.While nitric oxide synthase 1(NOS1)is recognized for its important in-volvement in various cancers,its specific function in ESCA remains unclear.AIM To explore the potential role and underlying mechanisms of NOS1 in ESCA.METHODS Survival rates were analyzed using GeneCards and Gene Expression Profiling Interactive Analysis.The effects and mechanisms of NOS1 on ESCA cells were evaluated via the Cell Counting Kit-8 assay,scratch assay,Transwell assay,flow cytometry,quantitative polymerase chain reaction,western blotting,and terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick end labeling staining.The protein interaction network was used to screen the interacting proteins of NOS1 and validate these interactions through co-immuno-precipitation and dual luciferase assays.Additionally,a nude mouse xenograft model was established to evaluate the effect of NOS1 in vivo.RESULTS The survival rate of patients with ESCA with high NOS1 expression was higher than that of patients with low NOS1 expression.NOS1 expression in ESCA cell lines was lower than that in normal esophageal epithelial cells.Overexpression of NOS1(oe-NOS1)inhibited proliferation,invasion,and migration abilities in ESCA cell lines,resulting in decreased autophagy levels and increased apoptosis,pyroptosis,and ferroptosis.Protein interaction studies confirmed the interaction between NOS1 and NOS1 adaptor protein(NOS1AP).Following oe-NOS1 and the silencing of NOS1AP,levels of P62 and microtubule-associated protein 1 light chain 3 beta increased both in vitro and in vivo.Furthermore,the expression levels of E-cadherin,along with the activation of phosphatidylinositol 3-kinase(PI3K)and protein kinase B(AKT),were inhibited in ESCA cell lines.CONCLUSION NOS1 and NOS1 proteins interact to suppress autophagy,activate the PI3K/AKT pathway,and exert anti-cancer effects in ESCA.展开更多
Terpenoids,one of the most diverse and structurally varied natural products in nature,are widely distributed in plants,microbes,and other organisms.Their structural diversity confers significant importance in medicine...Terpenoids,one of the most diverse and structurally varied natural products in nature,are widely distributed in plants,microbes,and other organisms.Their structural diversity confers significant importance in medicine,food,flavorings,and energy.However,traditional methods of plant extraction and chemical synthesis have limitations in industrial applications.Consequently,microbial cell factories have emerged as an important platform for terpenoid production.Terpene synthases(TPSs)are crucial in determining the structural and functional diversity of terpenoids.This review discussed the origin and classificationof TPSs,outlines commonly used TPS mining methods,and summarizes advances in TPS engineering.In addition,it also explores the influenceof machine learning on enzyme mining,the existing challenges and the future opportunities alongside cutting-edge technologies.展开更多
Isochorismate synthase(ICS),a key rate-limiting enzyme in the salicylic acid(SA)biosynthesis pathway in plants,is essential for plant growth and defense against diseases.However,there has been no report on ICS in suga...Isochorismate synthase(ICS),a key rate-limiting enzyme in the salicylic acid(SA)biosynthesis pathway in plants,is essential for plant growth and defense against diseases.However,there has been no report on ICS in sugarcane(Saccharum spp.).In this study,18 SsICSs,42 ShICSs,and 36 SzICSs were identified from the genomes of sugarcane AP85-441(Saccharum spontaneum),XTT22(Saccharum spp.hybrid cultivar),and ZZ1(Saccharum spp.hybrid cultivar),respectively.These were phylogenetically divided into three groups,forming distinct clades that were evolutionarily divergent from those in dicotyledonous species.The evolutionary profile of the ICS gene family suggested expansion through whole-genome duplication/segmental events and strong purifying selection.Promoter cis-element and transcriptome analyses indicated that the ICS gene family responded to disease stress.We cloned the ScICS(isochorismate synthase)gene from sugarcane cultivar XTT22 leaves,and found it was localized in chloroplasts.In vivo and in vitro interaction studies revealed an interaction between ScICS and an ScMYB transcription factor.We showed that ScWRKY28 positively regulated ScICS expression by binding to its promoter.ScICS overexpression in transgenic tobacco confirmed its effectiveness in enhancing disease resistance.There was a significant increase in SA content following pathogen infection along with activation of downstream signaling pathways and defense mechanisms.This study establishes the groundwork for functional studies of sugarcane ICS genes and enhances our understanding of the mechanisms of disease resistance in sugarcane.展开更多
African swine fever virus(ASFV)pB318L is an important protein for viral replication that acts as a membrane-bound trans-geranylgeranyl-diphosphate synthase(GGPPS)catalyzing the condensation of isopentenyl diphosphate(...African swine fever virus(ASFV)pB318L is an important protein for viral replication that acts as a membrane-bound trans-geranylgeranyl-diphosphate synthase(GGPPS)catalyzing the condensation of isopentenyl diphosphate(IPP)with allylic diphosphates.Recently we solved the crystal structure pB318L lacking N-terminal transmembrane region and performed a preliminary structural analysis.In this study,structure-based mutagenesis study and geranylgeranyl pyrophosphate(GGPP)production assay further revealed the key residues for the GGPPS activity.Structural comparison showed pB318L displays a strong similarity to typical GGPPSs instead of protein prenyltransferases.The phylogenetic analysis indicated pB318L may share a common ancestor with the GGPPSs from Brassicaceae plants rather than from its natural host.The subcellular localization analysis showed pB318L is localized in both nucleus and cytoplasm(including the endoplasmic reticulum membrane and mitochondria outer membrane).A unique N-terminal nuclear localization signal(NLS)following the transmembrane region was discovered in pB318L and the NLS was confirmed to be required for the nuclear import.We further revealed the NLS plays an essential role in the interaction with nuclear transporter karyopherin subunit alpha 1(KPNA1).Their interaction may suppress signal transducers and activators of transcription 1(STAT1)translocation and subsequently competitively inhibit nuclear import of IFNstimulated gene factor 3(ISGF3)complex.Our biochemical,structural and cellular analyses provide novel insights to pB318L that acts as an essential GGPPS that promotes viral replication and as a nuclear import protein that may be involved in immune evasion of ASFV.展开更多
OBJECTIVE:To explore the therapeutic effect and target of atractylenolide I(AT-I)on post-infectious irritable bowel syndrome(PI-IBS)rats.METHODS:Therefore,the preliminarily mechanism of AT-I in anti-PI-IBS were first ...OBJECTIVE:To explore the therapeutic effect and target of atractylenolide I(AT-I)on post-infectious irritable bowel syndrome(PI-IBS)rats.METHODS:Therefore,the preliminarily mechanism of AT-I in anti-PI-IBS were first predicted by network pharmacology and molecular docking,then the possible signaling pathways were systematically analyzed.Finally,the potential therapeutic targets and possible signaling pathways of AT-I on PI-IBS in Sprague-Dawley(SD)rat model were verified by experiments.RESULTS:AT-I could alleviate PI-IBS symptoms and reduce the expression of tumor necrosis factorα,interleukin-6 and Interferon-gamma in PI-IBS SD rat model and inhibit the c-Jun N-terminal kinase/inducible nitric oxide synthase(JNK/iNOS)pathway.Notably,AT-I treatment could inhibit the overexpression of polymeraseⅠand transcript release factor(PTRF).CONCLUSION:AT-I could alleviate PI-IBS symptoms through downregulation of PTRF and inhibiting the JNK/iNOS pathway.This study not only provides a scientific basis to clarify the anti-PI-IBS effect of AT-I and its mechanism but also suggests a novel promising therapeutic strategy to treat the PI-IBS.展开更多
During the course of mungbean (Phaseolus radiatus L.) germination, the rate of ethylene production and the activity of ACC synthase (1_aminocyclopropane_1_carboxylic acid synthase, EC4.4.1.4) began to increase in the ...During the course of mungbean (Phaseolus radiatus L.) germination, the rate of ethylene production and the activity of ACC synthase (1_aminocyclopropane_1_carboxylic acid synthase, EC4.4.1.4) began to increase in the 5th day of germination, and reached its peak in the 10th day and then decreased. The ethylene production and the activity of ACC synthase were obviously promoted by 10 μmol/L exogenous IAA (indole_3_acetic acid). The production of superoxide radical (O -· 2) and hydrogen peroxide (H 2O 2) were also promoted by exogenous IAA, suggesting that there was some relationship between active oxygen production and the activity of ACC synthase induced by exogenous IAA. The production of ethylene and the activity of ACC synthase increased dramatically when the seedlings were treated with exogenous O -· 2, whereas the exogenous H 2O 2 had no effects on the production of ethylene and the activity of ACC synthase. Exogenous SOD (superoxide dismutase, one scavenger of O -· 2) could inhibit the production of ethylene and the activity of ACC synthase, but exogenous CAT (catalase) could not. So it was possible that IAA would stimulate the activity of ACC synthase by inducing the production of O -· 2 in germinating mungbean seedlings, and this might be one of the regulating mechanism of ethylene synthesis in higher plants; the production of H 2O 2 induced by IAA was not the cause of the increase of the activity of ACC synthase and the production of ethylene.展开更多
基金Guangdong Basic and Applied Basic Research Foundation(2023B1515120050,2023A1515110529 and 2024A1515012454)Shenzhen Science and Technology Program(JCYJ20230807145759008 and KJZD20240903100206009).
文摘Acetolactate synthase(ALS)-targeting herbicides are among the most widely used weed-control chemicals globally.Mutations in the ALS gene can confer herbicide resistance in crops,thereby allowing selective elimination of weeds without harming crops.Herbicide-resistant ALS alleles were initially discovered in weeds and subsequently developed through artificial mutagenesis techniques.With the advancement of CRISPR/Cas technologies,various genome-editing tools are now available to introduce these resistant alleles,as well as novel variants,into diverse crop species.Moreover,emerging methodologies,such as directed evolution,enable the generation and screening of large populations of random ALS mutants.Consequently,ALS has become one of the most extensively targeted genes in plant gene evolution.This paper provides a comprehensive overview of both conventional and recently developed strategies for ALS evolution,with particular emphasis on CRISPR/Cas-based genome editing and directed evolution.Future perspectives on technological application are also discussed.By advancing our understanding of herbicide-resistant ALS allele development for crop improvement,these methodologies may also pave the way for their application to the evolution of other agronomically important genes.
基金financially supported in part by grants from Biological Breeding-National Science and Technology Major Project (2023ZD04040)Jiangsu Key Research and Development Program (BE2022384)Collaborative Innovation Center for Modern Crop Production co-sponsored by Province and Ministry (CICMCP) (No. 10)
文摘Carotenoids are natural pigments that are widely distributed in the flowers,fruits,and seeds of many plant species.These compounds not only endow diverse colors but also exhibit antioxidant,immune-modulatory,anti-aging,and photoprotective properties.Although carotenoid metabolism has been studied extensively in microbial and plant science,the genetic mechanisms underlying carotenoid metabolism in cotton remain underexplored.Here,we isolated gene GbDYA that regulates a dark-yellow anther color by map-based cloning using a BC1F1 population derived from a cross of Gossypium barbadense acc.Hai7124 with dark-yellow anthers and G.hirsutum acc.TM-1 with light-yellow anthers backcrossed with TM-1.GbDYA encodes phytoene synthase,a key rate-limiting enzyme in the carotenoid biosynthesis pathway.A long terminal repeat retrotransposon in the first exon of GhDYA(an ortholog of GbDYA in G.hirsutum acc.TM-1)caused loss of function and led to the light-yellow anther color.GbDYA is predominantly expressed in the early stages of anther development.Transcriptome,RT-qPCR and KEGG enrichment analyses revealed that GbDYA influences the synthesis and accumulation of carotenoids in anthers by modulating expression of key genes in the carotenoid biosynthesis pathway.Integrated transcriptomic and metabolomic analyses indicated that the accumulation of lutein,violaxanthin,antherxanthin,cryptoxanthin,zeaxanthin,andβ-carotene contributed to yellow coloration of anthers.Dual-luciferase and yeast one-hybrid assays confirmed that transcription factor GbMYB105(GB_A11G3511)binds to the promoter of GbDYA and activates its expression.High-temperature stress treatment indicated that carotenoids accumulation in anthers enhances pollen antioxidant activity.This study unravels the role of GbDYA in conferring the anther coloration,and provides the potential utilization by modulating accumulation of carotenoids in anthers to enhance pollen viability in high-temperature tolerance breeding in cotton.
基金supported by grants from the National Key Research and Development Program of China,No.2017YFA0105400(to LR)the Key Research and Development Program of Guangdong Province,No.2019B020236002(to LR)the National Natural Science Foundation of China,Nos.81972111(to LZ),81772349(to BL).
文摘Microglia,the resident monocyte of the central nervous system,play a crucial role in the response to spinal cord injury.However,the precise mechanism remains unclear.To investigate the molecular mechanisms by which microglia regulate the neuroinflammatory response to spinal cord injury,we performed single-cell RNA sequencing dataset analysis,focusing on changes in microglial subpopulations.We found that the MG1 subpopulation emerged in the acute/subacute phase of spinal cord injury and expressed genes related to cell pyroptosis,sphingomyelin metabolism,and neuroinflammation at high levels.Subsequently,we established a mouse model of contusive injury and performed intrathecal injection of siRNA and molecular inhibitors to validate the role of ceramide synthase 5 in the neuroinflammatory responses and pyroptosis after spinal cord injury.Finally,we established a PC12-BV2 cell co-culture system and found that ceramide synthase 5 and pyroptosis-associated proteins were highly expressed to induce the apoptosis of neuron cells.Inhibiting ceramide synthase 5 expression in a mouse model of spinal cord injury effectively reduced pyroptosis.Furthermore,ceramide synthase 5-induced pyroptosis was dependent on activation of the NLRP3 signaling pathway.Inhibiting ceramide synthase 5 expression in microglia in vivo reduced neuronal apoptosis and promoted recovery of neurological function.Pla2g7 formed a“bridge”between sphingolipid metabolism and ceramide synthase 5-mediated cell death by inhibiting the NLRP3 signaling pathway.Collectively,these findings suggest that inhibiting ceramide synthase 5 expression in microglia after spinal cord injury effectively suppressed microglial pyroptosis mediated by NLRP3,thereby exerting neuroprotective effects.
基金This work was supported by the National Natural Science Foundation of China(grant numbers 81560301 and 81160012)the Natural Science Foundation of Qinghai Province(grant number 2022-ZJ-905)‘2022 Qinghai Province Kunlun Talents High-end Innovation and Entrepreneurship Talents’Outstanding Talent Project.
文摘Background:Under hypoxia,exaggerated compensatory responses may lead to acute mountain sickness.The excessive vasodilatory effect of nitric oxide(NO)can lower the hypoxic pulmonary vasoconstriction(HPV)and peripheral blood pressure.While NO is catalyzed by various nitric oxide synthase(NOS)isoforms,the regulatory roles of these types in the hemodynamics of pulmonary and systemic circulation in living hypoxic animals remain unclear.Therefore,this study aims to investigate the regu-latory effects of different NOS isoforms on pulmonary and systemic circulation in hypoxic rats by employing selective NOS inhibitors and continuously monitoring hemodynamic parameters of both pulmonary and systemic circulation.Methods:Forty healthy male Sprague–Dawley(SD)rats were randomly divided into four groups:Control group(NG-nitro-D-arginine methyl ester,D-NAME),L-NAME group(non-selective NOS inhibitor,NG-nitro-L-arginine methyl ester),AG group(in-ducible NOS inhibitor group,aminoguanidine),and 7-NI group(neurological NOS in-hibitor,7-nitroindazole).Hemodynamic parameters of rats were monitored for 10 min after inhibitor administration and 5 min after induction of hypoxia[15%O2,2200 m a.sl.,582 mmHg(76.5 kPa),Xining,China]using the real-time dynamic monitoring model for pulmonary and systemic circulation hemodynamics in vivo.Serum NO concentra-tions and blood gas analysis were measured.Results:Under normoxia,mean arterial pressure and total peripheral vascular resist-ance were increased,and ascending aortic blood flow and serum NO concentration were decreased in the L-NAME and AG groups.During hypoxia,pulmonary arterial pressure and pulmonary vascular resistance were significantly increased in the L-NAME and AG groups.Conclusions:This compensatory mechanism activated by inducible NOS and en-dothelial NOS effectively counteracts the pulmonary hemodynamic changes induced by hypoxic stress.It plays a crucial role in alleviating hypoxia-induced pulmonary arte-rial hypertension.
基金Supported by a grant from the National Natural Science Foundation of China(no.82174457)。
文摘Background:Lung cancer is one of the deadliest cancers worldwide,creating a pressing need to develop novel drugs that inhibit oncogenic signaling pathways.Numerous studies have shown that berberine(BBR)has anti–lung cancer potential.We aimed to explore the anti–lung cancer effect of BBR and related mechanisms by targeting the glycogen synthase kinase 3β(GSK3β)/β-catenin pathway.Methods:Lung adenocarcinoma(LUAD)cells A549 and NCI-H1975 were treated with BBR.Results:Our results showed that BBR inhibited cell proliferation by decreasing c-Myc levels and induced cel cycle arrest in the G0/G1 phase by lowering cyclin D1 levels.BBR induced apoptosis by upregulating cleaved caspase 3 levels.BBR inhibited cell migration and invasion by decreasing N-cadherin levels.Furthermore,BBR upregulated the expression of GSK3βprotein and phospho-β-catenin proteins in the cytoplasm,while decreasing the expression ofβ-catenin protein.Next,LUAD cel s were exposed to CHIR-99021(a GSK3βinhibitor).This treatment led to an increase in c-Myc,cyclin D1,andβ-catenin levels at specific concentrations.BBR partially reversed the effects of CHIR-99021.Finally,LUAD cells were treated with CHIR-99021(4μmoL/L)combined with BBR(30 and 60μmoL/L)for 24 h.The expression of programmed death ligand 1(PD-L1)was assessed by Western blot analysis.Jurkat T cells and A549 cel s were cocultured for 24 h to examine the lactate dehydrogenase release rate.Results suggested that BBR suppressed the expression of PD-L1 and heightened the immune lethality of T cells.Conclusions:BBR suppressed the proliferative activity of LUAD cell lines A549 and NCI-H1975 in vitro,induced cell cycle arrest and cancer cel apoptosis in the G0/G1 stage,and repressed the migration and invasion of cancer cells.BBR reduced the PD-L1 protein expression and enhanced T-cell–mediated cytotoxicity.These effects appear to be related to BBR's regulation of the GSK3β/β-catenin pathway.
文摘[Objective]To confirm the function of the farnesyl diphosphate(FPP)cyclase encoded by orf2064 in Streptomyces exfoliatus UC5319.[Methods]orf2064 was expressed in Escherichia coli,and the recombinant protein was purified and assayed with FPP as the substrate.The reaction products were detected by GC-MS.An FPP-overproducing E.coli strain was engineered for heterologous expression of orf2064.The fermentation products were analyzed by GC-MS,and the target compound was isolated and structurally characterized by nuclear magnetic resonance spectroscopy(NMR).In addition,orf2064 was heterologously expressed in Streptomyces,and the fermentation products were analyzed by GC-MS.[Results]GC-MS revealed that both the in vitro reaction of the recombinant protein ORF2064 and the heterologous expression products in E.coli and Streptomyces consistently produced a compound with identical retention time and[M+]of m/z 204.Subsequent isolation,purification,and NMR analysis confirmed this compound as calarene.[Conclusion]The FPP cyclase encoded by orf2064 in S.exfoliatus is identified as an calarene synthase.
文摘Isoflavones which mainly distributed in leguminous plants have plenty of health benefits.Isoflavone synthase(IFS)is a membrane-associated cytochrome P450 enzyme(CYP450)which carries out the unique aryl-ring migration and hydroxylation.So far,few crystal structures of plant P450s have been obtained.We determined the crystal structure of IFS from Medicago truncatula at 1.9 by MAD method using a selenomethionine substituted crystal and conducted molecular docking and mutagenesis study.The structure of IFS complexed with imidazole exhibits the helix Iα-loop-helix Iβmotif which corresponds to helix I of other P 450s.Compared with structures of common P450s,IFS/imidazole structure contains an extra domain,i.e.,theγ-domain.The structure reveals a homodimer in which theγ-domain of one molecule interacts with theβ-domain of another.The plane of heme group makes an angle of approximately 40°with the helix Iα-loop-helix Iβmotif.Molecular docking combined with mutagenesis study suggested that Trp-128 and Asp-300 might play important roles in substrate binding and recognition.Phe-301,Ser-303 and Gly-305 from the helix Iα-loop-helix Iβmotif may play important roles in the aryl-ring migration.These novel structural features reveal insights into the unique reaction mechanism of IFS and provide a basis for engineering IFS in leguminous crops for health purpose.
基金supported by the National Natural Science Foundation of China(32161133010)the National Key Research and Development Program of China(2022YFD1700200)+2 种基金the Innovation Program of Chinese Academy of Agricultural Sciences(CAAS-CSCB-202302)the Shenzhen Science and Technology Program,China(KQTD20180411143628272)the Special Funds for Science Technology Innovation and Industrial Development of Shenzhen Dapeng New District,China(PT202101-02).
文摘Chitin is an abundant natural biopolymer that plays a crucial role in insect growth and development as a fundamental structural component of the exoskeleton.The membrane-integralβ-glycosyltransferase,chitin synthase,has been identified as the central component in chitin biosynthesis.However,the precise roles of other proteins in facilitating chitin synthase in chitin biosynthesis remain unclear.In this study,we employed split-ubiquitin membrane yeast two-hybrid(MYTH)and pull-down assays to demonstrate the physical interaction between Twinstar(Tsr),a small molecular protein in the actin-depolymerizing factor ADF/Cofilin protein family,and chitin synthase Krotzkopf verkehrt(Kkv)in Drosophila melanogaster in vitro.The RNA interference(RNAi)-mediated global knockdown of Tsr in D.melanogaster resulted in larval lethality.Furthermore,targeted suppression of Tsr in the tracheal and epidermal tissues also led to larval mortality,while knocking down Tsr in the wing tissues led to wrinkled wings.Additionally,silencing Tsr not only reduced the chitin content in the first longitudinal vein of the wings but also led to the absence of the chitin lamellar structure.To validate the functional conservation of Tsr in other insect orders,the two agricultural pests Ostrinia furnacalis and Tribolium castaneum,representing lepidoptera and coleoptera insects,respectively,were investigated.Knockdown experiments targeting the Drosophila Tsr orthologues OfTsr in O.furnacalis and TcTsr in T.castaneum produced abnormal larvae during molting or pupation in O.furnacalis and lethality in T.castaneum.Our findings not only improve ourknowledge of the chitin biosynthesis machinery in insect cuticles but also provide new potential targets for the control of major agricultural pests.
基金Supported by the National Natural Science Foundation of China,No.81000201.
文摘BACKGROUND Esophageal cancer(ESCA)is among the most prevalent and lethal tumors globally.While nitric oxide synthase 1(NOS1)is recognized for its important in-volvement in various cancers,its specific function in ESCA remains unclear.AIM To explore the potential role and underlying mechanisms of NOS1 in ESCA.METHODS Survival rates were analyzed using GeneCards and Gene Expression Profiling Interactive Analysis.The effects and mechanisms of NOS1 on ESCA cells were evaluated via the Cell Counting Kit-8 assay,scratch assay,Transwell assay,flow cytometry,quantitative polymerase chain reaction,western blotting,and terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick end labeling staining.The protein interaction network was used to screen the interacting proteins of NOS1 and validate these interactions through co-immuno-precipitation and dual luciferase assays.Additionally,a nude mouse xenograft model was established to evaluate the effect of NOS1 in vivo.RESULTS The survival rate of patients with ESCA with high NOS1 expression was higher than that of patients with low NOS1 expression.NOS1 expression in ESCA cell lines was lower than that in normal esophageal epithelial cells.Overexpression of NOS1(oe-NOS1)inhibited proliferation,invasion,and migration abilities in ESCA cell lines,resulting in decreased autophagy levels and increased apoptosis,pyroptosis,and ferroptosis.Protein interaction studies confirmed the interaction between NOS1 and NOS1 adaptor protein(NOS1AP).Following oe-NOS1 and the silencing of NOS1AP,levels of P62 and microtubule-associated protein 1 light chain 3 beta increased both in vitro and in vivo.Furthermore,the expression levels of E-cadherin,along with the activation of phosphatidylinositol 3-kinase(PI3K)and protein kinase B(AKT),were inhibited in ESCA cell lines.CONCLUSION NOS1 and NOS1 proteins interact to suppress autophagy,activate the PI3K/AKT pathway,and exert anti-cancer effects in ESCA.
基金supported by the National Key Research and Development Program of China(2020YFA0908300)the Natural Science Foundation of China(22138006,22278240).
文摘Terpenoids,one of the most diverse and structurally varied natural products in nature,are widely distributed in plants,microbes,and other organisms.Their structural diversity confers significant importance in medicine,food,flavorings,and energy.However,traditional methods of plant extraction and chemical synthesis have limitations in industrial applications.Consequently,microbial cell factories have emerged as an important platform for terpenoid production.Terpene synthases(TPSs)are crucial in determining the structural and functional diversity of terpenoids.This review discussed the origin and classificationof TPSs,outlines commonly used TPS mining methods,and summarizes advances in TPS engineering.In addition,it also explores the influenceof machine learning on enzyme mining,the existing challenges and the future opportunities alongside cutting-edge technologies.
基金supported by the National Natural Science Foundation of China(32472166,32172063,and 31771861)the Innovative Team Construction Project of the Modern Agricultural Industry Technology System in Guangdong Province by Agricultural Product Units(Sugarcane and Sisal Industry Technology System,2024CXTD03-06)South China Agricultural University Students Innovation and Entrepreneurship Training Program(2024105641195)。
文摘Isochorismate synthase(ICS),a key rate-limiting enzyme in the salicylic acid(SA)biosynthesis pathway in plants,is essential for plant growth and defense against diseases.However,there has been no report on ICS in sugarcane(Saccharum spp.).In this study,18 SsICSs,42 ShICSs,and 36 SzICSs were identified from the genomes of sugarcane AP85-441(Saccharum spontaneum),XTT22(Saccharum spp.hybrid cultivar),and ZZ1(Saccharum spp.hybrid cultivar),respectively.These were phylogenetically divided into three groups,forming distinct clades that were evolutionarily divergent from those in dicotyledonous species.The evolutionary profile of the ICS gene family suggested expansion through whole-genome duplication/segmental events and strong purifying selection.Promoter cis-element and transcriptome analyses indicated that the ICS gene family responded to disease stress.We cloned the ScICS(isochorismate synthase)gene from sugarcane cultivar XTT22 leaves,and found it was localized in chloroplasts.In vivo and in vitro interaction studies revealed an interaction between ScICS and an ScMYB transcription factor.We showed that ScWRKY28 positively regulated ScICS expression by binding to its promoter.ScICS overexpression in transgenic tobacco confirmed its effectiveness in enhancing disease resistance.There was a significant increase in SA content following pathogen infection along with activation of downstream signaling pathways and defense mechanisms.This study establishes the groundwork for functional studies of sugarcane ICS genes and enhances our understanding of the mechanisms of disease resistance in sugarcane.
基金supported by the grants from the National Natural Science Foundation of China to Heng Zhang(31970152)the Strategic Priority Research Program of CAS to Yu-Hui Dong(XDB37040302)the CAS Emergency Research Project on African Swine Fever to Yu-Hui Dong(Grant KJZDSW-L06-01).
文摘African swine fever virus(ASFV)pB318L is an important protein for viral replication that acts as a membrane-bound trans-geranylgeranyl-diphosphate synthase(GGPPS)catalyzing the condensation of isopentenyl diphosphate(IPP)with allylic diphosphates.Recently we solved the crystal structure pB318L lacking N-terminal transmembrane region and performed a preliminary structural analysis.In this study,structure-based mutagenesis study and geranylgeranyl pyrophosphate(GGPP)production assay further revealed the key residues for the GGPPS activity.Structural comparison showed pB318L displays a strong similarity to typical GGPPSs instead of protein prenyltransferases.The phylogenetic analysis indicated pB318L may share a common ancestor with the GGPPSs from Brassicaceae plants rather than from its natural host.The subcellular localization analysis showed pB318L is localized in both nucleus and cytoplasm(including the endoplasmic reticulum membrane and mitochondria outer membrane).A unique N-terminal nuclear localization signal(NLS)following the transmembrane region was discovered in pB318L and the NLS was confirmed to be required for the nuclear import.We further revealed the NLS plays an essential role in the interaction with nuclear transporter karyopherin subunit alpha 1(KPNA1).Their interaction may suppress signal transducers and activators of transcription 1(STAT1)translocation and subsequently competitively inhibit nuclear import of IFNstimulated gene factor 3(ISGF3)complex.Our biochemical,structural and cellular analyses provide novel insights to pB318L that acts as an essential GGPPS that promotes viral replication and as a nuclear import protein that may be involved in immune evasion of ASFV.
基金The University Collaborative Innovation Project of Anhui:Creation of a Combined Animal Model of Coronary Heart Disease based on the Theory of Xin'an Medicine(No.GXXT-2020-024)Start-up Funding for Doctoral Research at Wannan Medical College(WYRCQD2018009)Horizontal Project of South Anhui Medical College(H202003)。
文摘OBJECTIVE:To explore the therapeutic effect and target of atractylenolide I(AT-I)on post-infectious irritable bowel syndrome(PI-IBS)rats.METHODS:Therefore,the preliminarily mechanism of AT-I in anti-PI-IBS were first predicted by network pharmacology and molecular docking,then the possible signaling pathways were systematically analyzed.Finally,the potential therapeutic targets and possible signaling pathways of AT-I on PI-IBS in Sprague-Dawley(SD)rat model were verified by experiments.RESULTS:AT-I could alleviate PI-IBS symptoms and reduce the expression of tumor necrosis factorα,interleukin-6 and Interferon-gamma in PI-IBS SD rat model and inhibit the c-Jun N-terminal kinase/inducible nitric oxide synthase(JNK/iNOS)pathway.Notably,AT-I treatment could inhibit the overexpression of polymeraseⅠand transcript release factor(PTRF).CONCLUSION:AT-I could alleviate PI-IBS symptoms through downregulation of PTRF and inhibiting the JNK/iNOS pathway.This study not only provides a scientific basis to clarify the anti-PI-IBS effect of AT-I and its mechanism but also suggests a novel promising therapeutic strategy to treat the PI-IBS.
文摘During the course of mungbean (Phaseolus radiatus L.) germination, the rate of ethylene production and the activity of ACC synthase (1_aminocyclopropane_1_carboxylic acid synthase, EC4.4.1.4) began to increase in the 5th day of germination, and reached its peak in the 10th day and then decreased. The ethylene production and the activity of ACC synthase were obviously promoted by 10 μmol/L exogenous IAA (indole_3_acetic acid). The production of superoxide radical (O -· 2) and hydrogen peroxide (H 2O 2) were also promoted by exogenous IAA, suggesting that there was some relationship between active oxygen production and the activity of ACC synthase induced by exogenous IAA. The production of ethylene and the activity of ACC synthase increased dramatically when the seedlings were treated with exogenous O -· 2, whereas the exogenous H 2O 2 had no effects on the production of ethylene and the activity of ACC synthase. Exogenous SOD (superoxide dismutase, one scavenger of O -· 2) could inhibit the production of ethylene and the activity of ACC synthase, but exogenous CAT (catalase) could not. So it was possible that IAA would stimulate the activity of ACC synthase by inducing the production of O -· 2 in germinating mungbean seedlings, and this might be one of the regulating mechanism of ethylene synthesis in higher plants; the production of H 2O 2 induced by IAA was not the cause of the increase of the activity of ACC synthase and the production of ethylene.
