The effects of mepiquat chloride(DPC)on the Cry1Ac protein content in Bacillus thuringiensis(Bt)cotton boll shells under high temperature and drought stress were investigated to provide a theoretical reference for Bt ...The effects of mepiquat chloride(DPC)on the Cry1Ac protein content in Bacillus thuringiensis(Bt)cotton boll shells under high temperature and drought stress were investigated to provide a theoretical reference for Bt cotton breeding and high-yield and-efficiency cotton cultivation.This study was conducted using Bt cotton cultivar‘Sikang 3'during the 2020 and 2021 growing seasons at Yangzhou University Farm,Yangzhou,Jiangsu Province,China.Potted cotton plants were exposed to high temperature and drought stress,and sprayed with either 20 mg L^(-1)DPC or water(CK).Seven days after treatment,the Cry1Ac protein content,α-ketoglutarate content,pyruvic acid content,glutamate synthase activity,glutamic oxaloacetic transaminase activity,soluble protein content,and amino acid content were measured,and transcriptome sequencing was performed.DESeq was used for differential gene analysis.Under the DPC treatment,the Cry1Ac protein content increased by 4.7-11.9% compared to CK.Theα-ketoglutarate content,pyruvic acid content,glutamate synthase activity,glutamic oxaloacetic transaminase activity,soluble protein content,and amino acid content all increased.Transcriptome analysis revealed 7,542 upregulated genes and 10,449 downregulated genes for DPC vs.CK.Gene ontology(GO)and Kyoto Encyclopedia of Gene and Genomes(KEGG)analyses showed that the differentially expressed genes were mainly involved in biological processes,such as carbon and amino acid metabolism.For example,genes encoding 6-phosphofructokinase,pyruvate kinase,glutamic pyruvate transaminase,pyruvate dehydrogenase,citrate synthase,isocitrate dehydrogenase,2-oxoglutarate dehydrogenase,glutamate synthase,1-pyrroline-5-carboxylate dehydrogenase,glutamic oxaloacetic transaminase,amino-acid N-acetyltransferase,and acetylornithine deacetylase were all significantly upregulated.The DPC treatment increased pyruvate,α-ketoglutarate,and oxaloacetate by increasing the operational rate of the glycolytic pathway of the citric acid cycle.It also significantly upregulated the genes encoding glutamate synthase,pyrrolidine-5-carboxylic acid dehydrogenase,glutamate oxaloacetate transaminase,and N-acetylglutamate synthetase,while it downregulated the genes encoding glutamine synthetase.Therefore,the synthesis of aspartic acid,glutamic acid,pyruvate,and arginine increased after treatment with DPC,and the Cry1Ac protein content was increased by regulating carbon and amino acid metabolism.展开更多
1-Aminocyclopropane-1-carboxylic acid （ACC） synthase （ACS） is the principal enzyme in phytohormone ethylene biosynthesis. Previous studies have shown that the hypervariable C-terminus of ACS is proteolytically pro...1-Aminocyclopropane-1-carboxylic acid （ACC） synthase （ACS） is the principal enzyme in phytohormone ethylene biosynthesis. Previous studies have shown that the hypervariable C-terminus of ACS is proteolytically processed in vivo. However, the protease responsible for this has not yet been identified. In the present study, we investigated the processing of the 55-kDa full-length tomato ACS （LeACS2） into 52-, 50- and 49-kDa truncated isoforms in ripening tomato （Lycopersicon esculentum Mill. cv. Cooperation 903） fruit using the sodium dodecyl sulfate-boiling method. Meanwhile, an LeACS2-processing protease was purified via multi-step column chromatography from tomato fruit. Subsequent biochemical analysis of the 64-kDa purified protease revealed that it is a metalloprotease active at multiple cleavage sites within the hypervariable C-terminus of LeACS2. N-terminal sequencing and matrix-assisted laser desorption/ionization time-of-flight analysis indicated that the LeACS2-processing metalloprotease cleaves at the C-terminal sites Lys^438, Glu^447, Lys^448, Asn^456, Ser^460, Ser^462, Lys^463, and Leu^474, but does not cleave the N- terminus of LeACS2. Four C-terminus-deleted （26-50 amino acids） LeACS2 fusion proteins were overproduced and subjected to proteolysis by this metalloprotease to identify the multiple cleavage sites located on the N-terminal side of the phosphorylation site Ser^460. The results indisputably confirmed the presence of cleavage sites within the region between the α-helix domain （H14） and Ser^460 for this metalloprotease. Furthermore, the resulting C-terminally truncated LeACS2 isoforms were active enzymatically. Because this protease could produce LeACS2 isoforms in vitro similar to those detected in vivo, it is proposed that this metalloprotease may be involved in the proteolysis of LeACS2 in vivo.展开更多
Inhibition ofβ-site amyloid precursor protein-cleaving enzyme 1(BACE1)or glycogen synthase kinase-3β(GSK-3β)is estimated to be the central therapeutic approach for Alzheimer’s disease(AD).In this study,water extra...Inhibition ofβ-site amyloid precursor protein-cleaving enzyme 1(BACE1)or glycogen synthase kinase-3β(GSK-3β)is estimated to be the central therapeutic approach for Alzheimer’s disease(AD).In this study,water extract of Kangenkaryu,its crude drug and chemical composition used in oriental medicine were evaluated regarding their BACE1 and GSK-3βinhibitory activities.Fluorescence resonance energy transfer was used to characterize the BACE1 inhibitory effect of Kangen-karyu,its crude drug and chemical composition.GSK-3βactivity was determined using the Kinase-Glo Luminescent Kinase Assay Platform.The water extract of Kangen-karyu inhibited BACE1 and GSK-3βin concentration-dependent manners when compared with reference drugs,quercetin and luteolin.Among six components of Kangen-karyu,the water extracts of Salviae Miltiorrhizae Radix or Cyperi Rhizoma exhibited significant inhibitory effects on BACE1 and GSK-3β.Among the constituents of Salviae Miltiorrhizae Radix extract,salvianolic acid C,salvianolic acid A,rosmarinic acid,and magnesium lithospermate B significantly inhibited BACE1.In addition,they inhibited GSK-3βwith an IC50 value range of 6.97 to 135.35μM.From these results,one of the effectiveness and its mechanisms of action of Kangen-karyu against AD may be the inhibition of BACE1 and GSK-3β,and one of the active ingredients of Kangen-karyu is Salviae Miltiorrhizae Radix and its constituents.展开更多
基金supported by the National Natural Science Foundation of China(31901462)the Natural Science Foundation of the Jiangsu Higher Education Institutions,China(22KJA210005)+1 种基金the Priority Academic Program Development of Jiangsu Higher Education Institutions,China(PAPD)the Brand Professional Construction Program of Jiangsu Higher Education Institutions,China。
文摘The effects of mepiquat chloride(DPC)on the Cry1Ac protein content in Bacillus thuringiensis(Bt)cotton boll shells under high temperature and drought stress were investigated to provide a theoretical reference for Bt cotton breeding and high-yield and-efficiency cotton cultivation.This study was conducted using Bt cotton cultivar‘Sikang 3'during the 2020 and 2021 growing seasons at Yangzhou University Farm,Yangzhou,Jiangsu Province,China.Potted cotton plants were exposed to high temperature and drought stress,and sprayed with either 20 mg L^(-1)DPC or water(CK).Seven days after treatment,the Cry1Ac protein content,α-ketoglutarate content,pyruvic acid content,glutamate synthase activity,glutamic oxaloacetic transaminase activity,soluble protein content,and amino acid content were measured,and transcriptome sequencing was performed.DESeq was used for differential gene analysis.Under the DPC treatment,the Cry1Ac protein content increased by 4.7-11.9% compared to CK.Theα-ketoglutarate content,pyruvic acid content,glutamate synthase activity,glutamic oxaloacetic transaminase activity,soluble protein content,and amino acid content all increased.Transcriptome analysis revealed 7,542 upregulated genes and 10,449 downregulated genes for DPC vs.CK.Gene ontology(GO)and Kyoto Encyclopedia of Gene and Genomes(KEGG)analyses showed that the differentially expressed genes were mainly involved in biological processes,such as carbon and amino acid metabolism.For example,genes encoding 6-phosphofructokinase,pyruvate kinase,glutamic pyruvate transaminase,pyruvate dehydrogenase,citrate synthase,isocitrate dehydrogenase,2-oxoglutarate dehydrogenase,glutamate synthase,1-pyrroline-5-carboxylate dehydrogenase,glutamic oxaloacetic transaminase,amino-acid N-acetyltransferase,and acetylornithine deacetylase were all significantly upregulated.The DPC treatment increased pyruvate,α-ketoglutarate,and oxaloacetate by increasing the operational rate of the glycolytic pathway of the citric acid cycle.It also significantly upregulated the genes encoding glutamate synthase,pyrrolidine-5-carboxylic acid dehydrogenase,glutamate oxaloacetate transaminase,and N-acetylglutamate synthetase,while it downregulated the genes encoding glutamine synthetase.Therefore,the synthesis of aspartic acid,glutamic acid,pyruvate,and arginine increased after treatment with DPC,and the Cry1Ac protein content was increased by regulating carbon and amino acid metabolism.
基金Supported by Hong Kong Research Grant Council （HKUST6102/02M and HKUST6105/01M） and the National Natural Science Foundation of China （30129001）.
文摘1-Aminocyclopropane-1-carboxylic acid （ACC） synthase （ACS） is the principal enzyme in phytohormone ethylene biosynthesis. Previous studies have shown that the hypervariable C-terminus of ACS is proteolytically processed in vivo. However, the protease responsible for this has not yet been identified. In the present study, we investigated the processing of the 55-kDa full-length tomato ACS （LeACS2） into 52-, 50- and 49-kDa truncated isoforms in ripening tomato （Lycopersicon esculentum Mill. cv. Cooperation 903） fruit using the sodium dodecyl sulfate-boiling method. Meanwhile, an LeACS2-processing protease was purified via multi-step column chromatography from tomato fruit. Subsequent biochemical analysis of the 64-kDa purified protease revealed that it is a metalloprotease active at multiple cleavage sites within the hypervariable C-terminus of LeACS2. N-terminal sequencing and matrix-assisted laser desorption/ionization time-of-flight analysis indicated that the LeACS2-processing metalloprotease cleaves at the C-terminal sites Lys^438, Glu^447, Lys^448, Asn^456, Ser^460, Ser^462, Lys^463, and Leu^474, but does not cleave the N- terminus of LeACS2. Four C-terminus-deleted （26-50 amino acids） LeACS2 fusion proteins were overproduced and subjected to proteolysis by this metalloprotease to identify the multiple cleavage sites located on the N-terminal side of the phosphorylation site Ser^460. The results indisputably confirmed the presence of cleavage sites within the region between the α-helix domain （H14） and Ser^460 for this metalloprotease. Furthermore, the resulting C-terminally truncated LeACS2 isoforms were active enzymatically. Because this protease could produce LeACS2 isoforms in vitro similar to those detected in vivo, it is proposed that this metalloprotease may be involved in the proteolysis of LeACS2 in vivo.
文摘Inhibition ofβ-site amyloid precursor protein-cleaving enzyme 1(BACE1)or glycogen synthase kinase-3β(GSK-3β)is estimated to be the central therapeutic approach for Alzheimer’s disease(AD).In this study,water extract of Kangenkaryu,its crude drug and chemical composition used in oriental medicine were evaluated regarding their BACE1 and GSK-3βinhibitory activities.Fluorescence resonance energy transfer was used to characterize the BACE1 inhibitory effect of Kangen-karyu,its crude drug and chemical composition.GSK-3βactivity was determined using the Kinase-Glo Luminescent Kinase Assay Platform.The water extract of Kangen-karyu inhibited BACE1 and GSK-3βin concentration-dependent manners when compared with reference drugs,quercetin and luteolin.Among six components of Kangen-karyu,the water extracts of Salviae Miltiorrhizae Radix or Cyperi Rhizoma exhibited significant inhibitory effects on BACE1 and GSK-3β.Among the constituents of Salviae Miltiorrhizae Radix extract,salvianolic acid C,salvianolic acid A,rosmarinic acid,and magnesium lithospermate B significantly inhibited BACE1.In addition,they inhibited GSK-3βwith an IC50 value range of 6.97 to 135.35μM.From these results,one of the effectiveness and its mechanisms of action of Kangen-karyu against AD may be the inhibition of BACE1 and GSK-3β,and one of the active ingredients of Kangen-karyu is Salviae Miltiorrhizae Radix and its constituents.
