The critical antioxidant catalase(CAT)breaks down hydrogen peroxide induced by environmental stresses.Here we cloned full length catalase cDNA from Lymantria dispar asiatic(LdCAT).Bioinformatic analyses showed that op...The critical antioxidant catalase(CAT)breaks down hydrogen peroxide induced by environmental stresses.Here we cloned full length catalase cDNA from Lymantria dispar asiatic(LdCAT).Bioinformatic analyses showed that open reading frames of LdCAT contains 1524 bp,encoding 507 amino acids with molecular weight of 126.99 kDa,theoretical pI of 5.00,aliphatic index of 29.92,grand average of hydropathicity of 0.764,and instability index(II)of 46.56.Protein BLAST and multiple sequence alignment indicated that LdCAT had high identity with CAT from other insects,especially lepidopterans.In a phylogenetic analysis,LdCAT was most similar to CAT from Spodoptera litura and S.exigua.Quantitative realtime polymerase chain reaction showed that LdCAT transcripts in all instar larvae and the five tissues tested,verifying the ubiquity of LdCAT in L.disapr.Moreover,LdCAT of third instar larvae was significantly upregulated after they fed on avermectin at sublethal and LC10 doses.The highest relative transcript levels were found 2 h after an avermectin spray at LC90,and in the cuticula,rather than heads,fat bodies,malpighian tubes,and midguts after a spray avermectin at a sublethal concentration.The expression level of LdCAT under pesticide stresses here suggested that CAT is an important antioxidant enzyme of L.disapr defensing against pesticide stress and may be a good target for controlling this pest.展开更多
The Guanine nucleotide exchange factor Vav2(Vav2) is a member of the Vav family that serves as an important regulators for the Rho family of Ras-related GTPases. In the current study, an ortholog(Lj-Vav2) of Vav2 was ...The Guanine nucleotide exchange factor Vav2(Vav2) is a member of the Vav family that serves as an important regulators for the Rho family of Ras-related GTPases. In the current study, an ortholog(Lj-Vav2) of Vav2 was identified in the lamprey(Lampetra japonica). To elucidate the phylogenetic relationship of Vav2, the metazoan genome databases were analyzed to mine the ortholog of Vav. It was found that Vav2 genes were only existed in vertebrates and Lj-Vav2 was the original one found in agnathans. The evolutionary dynamics of conserved motifs of Vav2 were explored using combined amino acid sequence as markers, and it is revealed that the Calponin homology(CH) domain, Dbl-homologous(DH) domain, Pleckstrin homology(PH) domain, Cysteine-rich(C1)domains, Src homology 3(SH3) domains and Src homology 2(SH2) domain were conserved throughout the Vav2 gene family in vertebrates during gene evolution. Relative quantitative real-time PCR analysis showed that the LjVav2 was distributed in the heart, kidney, supraneural myeloid body, liver, gill and lymphocyte-like cells. The LjVav2 was found to be expressed in these tissues, and the level of which was upregulated in lymphocyte-like cells after the animal was stimulated with LPS. These results indicated that the Lj-Vav2 might be involved in the immune response of lymphocyte-like cells in lamprey. Meanwhile, our findings provided a foundation for further investigation of the function of Lj-Vav2 in the primary vertebrate.展开更多
Sea Island cotton(Gossypium barbadense L.) has been highly valued in Verticillium wilt resistance and many fiber qualities including fiber length,strength,and fineness.To identify whether
Verticillium dahliae Kleb.is a necrotrophic plant pathogen which causes serious soil borne vascular disease in cotton.The molecular basis the defense response of cotton to this pathogen is
CAP,an adenylyl cyclase-associated protein,is predicted to be involved in cytoskeletal organization and signal transduction.Recently,we found that CAP may play an important role in fuzz-like fiber cell initiation in c...CAP,an adenylyl cyclase-associated protein,is predicted to be involved in cytoskeletal organization and signal transduction.Recently,we found that CAP may play an important role in fuzz-like fiber cell initiation in cotton.For the further research,we isolated two CAP homologues from wild展开更多
The zinc finger proteins belong to the largest family of transcription factors.But there is little research of Cys2/His2 type zinc finger proteins in cotton,and there is no submission of correlating
Studies on the cold-responsive genes and cold signaling of woody species drop far behind in comparison to herbaceous plants.Due to similar lignified structure,perennial characteristic,and enhanced tolerance,it seems m...Studies on the cold-responsive genes and cold signaling of woody species drop far behind in comparison to herbaceous plants.Due to similar lignified structure,perennial characteristic,and enhanced tolerance,it seems much easier to find strongly antifreeze genes and obtain effective results in transgenic woody plants.In this study,Ammopiptanthus mongolicus,an evergreen,broadleaf and cold-resist leguminous shrub growing in the desert of Inner Mongolia,was used as a material for low-temperature induced gene isolation.Through differential expression analysis induced by low-temperature,thirteen up-regulated cDNAs were identified.One of them,AmEBP1,(accession number:DQ519359)confers enhanced cold-tolerance to both transgenic E.coli and transgenic Arabidopsis.Results suggest that AmEBP1 can stimulate the synthesis of ribosome and the dephosphyration of the α-subunit of initiation factor 2(eIF2α),and subsequently promote the translation process.By which the transgenic plants obtained increased cold-resistant ability.展开更多
In this study, we report that we successfully cloned and sequenced a chitinase gene from the ovotestis of Kuroda’s sea hare Aplysia kurodai. By using reverse transcription-polymerase chain reaction (RT-PCR) and a sys...In this study, we report that we successfully cloned and sequenced a chitinase gene from the ovotestis of Kuroda’s sea hare Aplysia kurodai. By using reverse transcription-polymerase chain reaction (RT-PCR) and a system for the 5’ and 3’ rapid amplification of cDNA ends, we obtained a 1352 bp chitinase gene (AkChi) from the ovotestis of A. kurodai. AkChi contains a 1263 bp open reading frame that encodes 421 amino acids. The domain structure predicted from the deduced amino acid sequence was an N-terminal signal peptide and a catalytic domain of glycoside hydrolase (GH) family 18 chitinase. A comparative analysis of the deduced amino acid sequences of AkChi with those of the acidic mammalian chitinase of the California sea hare Aplysia californica revealed the highest homology at 83%. The purified chitinase from the ovotestis was digested by trypsin, and 119 residues of digested peptides were consistent with the deduced amino acid sequence of AkChi. We used RT-PCR to evaluate the expression of AkChi in various tissues of A. kurodai, and we observed that AkChi was expressed only in the ovotestis. A phylogenetic tree analysis, performed using the amino acid sequences of AkChi and known GH family 18 chitinases, showed that AkChi was separated from the molluscan chitinases with a chitin binding domain. To our knowledge, this is the first study demonstrating the cDNA cloning of an ovotestis chitinase from a sea hare.展开更多
Phenylalanine ammonia-lyase(PAL),which catalyzes the conversion from L-phenylalanine to trans-cinnamic acid,is a well-known key enzyme and a connecting step between primary and secondary metabolisms in the phenylpropa...Phenylalanine ammonia-lyase(PAL),which catalyzes the conversion from L-phenylalanine to trans-cinnamic acid,is a well-known key enzyme and a connecting step between primary and secondary metabolisms in the phenylpropanoid biosynthetic pathway of plants and microbes.Schisandra chinensis,a woody vine plant belonging to the family of Magnoliaceae,is a rich source of dibenzocyclooctadiene lignans exhibiting potent activity.However,the functional role of PAL in the biosynthesis of lignan is relatively limited,compared with those in lignin and flavonoids biosynthesis.Therefore,it is essential to clone and characterize the PAL genes from this valuable medicinal plant.In this study,molecular cloning and characterization of three PAL genes(ScPAL1−3)from S.chinensis was carried out.ScPALs were cloned using RACE PCR.The sequence analysis of the three ScPALs was carried out to give basic characteristics followed by docking analysis.In order to determine their catalytic activity,recombinant protein was obtained by heterologous expression in pCold-TF vector in Escherichia coli(BL21-DE3),followed by Ni-affinity purification.The catalytic product of the purified recombinant proteins was verified using RP-HPLC through comparing with standard compounds.The optimal temperature,pH value and effects of different metal ions were determined.Vmax,Kcat and Km values were determined under the optimal conditions.The expression of three ScPALs in different tissues was also determined.Our work provided essential information for the function of ScPALs.展开更多
A hexokinase gene named MdHXK1(MDP0000309677) was cloned from ‘Gala' apple(Malus × domestica Borkh.). Sequence analysis showed that the MdHXK1 gene was 1 497 bp long and encoded 499 amino acids. The predicte...A hexokinase gene named MdHXK1(MDP0000309677) was cloned from ‘Gala' apple(Malus × domestica Borkh.). Sequence analysis showed that the MdHXK1 gene was 1 497 bp long and encoded 499 amino acids. The predicted molecular mass of this protein was 54.05 kD, and the pI was 5.76. A phylogenetic tree indicated apple MdHXK1 exhibited the highest sequence similarity to Pyrus bretschneideri PbHXK1. Analysis of the functional domain showed that the MdHXK1 protein included two conserved kinase domains. The prediction of subcellular localization suggested that the MdHXK1 protein was mainly localized in the cytoplasm. There was an indication that MdHXK1 existed as one copy in the apple genome by Southern blotting. Silico analysis suggested that the promoter sequence contained several typical cis-acting elements, including defense, sugar signaling and phytohormone responsive elements. Quantitative real-time PCR analysis demonstrated that the MdHXK1 gene was mainly expressed in stem and flower tissues. During the development of apple fruits, the expression of the MdHXK1 gene initially increased and then decreased. The changes on Glc phosphorylation relative activity and glucose concentration showed the same trend. In addition, the expression of this gene was induced by salt stress, low temperature, and abscisic acid(ABA). Finally, we obtained and purified the fused MdHXK1 protein by recombinant prokaryotic expression. Studies have demonstrated that MdHXK1 may participate in sugar metabolism in apple fruits. Enzyme encoded by MdHXK1 is a key factor in the mediation of sugar accumulation. Recently,researchers on hexokinase at home and abroad mainly focused on model plants, such as Arabidopsis, tobacco and rice, but orchard fruit like apple were underresearched. Our research established the foundation for the further study of the functions of MdHXK1.展开更多
Objective To identlfy the member of the caspase famlly proteases involved in Y-radiation-inducedapoptosis in HL-60 cells and to study the expression or the caspase gene in normal, apoptotic cells and in immortal tumor...Objective To identlfy the member of the caspase famlly proteases involved in Y-radiation-inducedapoptosis in HL-60 cells and to study the expression or the caspase gene in normal, apoptotic cells and in immortal tumor cells. Methods By using degenerate oligonucleotide primers encoding the highly conserved peptides that were present in all known caspases, we performed RT-PCR on poly(A)RNA from γ-radiation-induced apoptotic HL-6o cells.Caspase-3 mRNA in apoptotic HL-6o cells and in human tumor cell lines was analyzed by Northern blot. Results The amplified DNA fragment was identiried with caspase-3 cDNA by cloning and sequencing. The Northern blot analysis of caspase-3 mRNA of different human tumor cell lines showed that the caspase-3 gene transcript was more highly expressed in leukemia cell lines and the SH-SY5Y cell line than in HeLa and MCF-7 cells. It was more highly expressed in the radiation-induced apoptotic HL-6o cells than in control HL-6o cells. Conclusion These results indicated that caspase-3 was involved in γ-radiation-induced apoptosis in HL-6o cells. The high level of expression or caspase-3 may aid efforts to understand the insensitivity of some tumor cells to radlation, their inherent ability to survive, and展开更多
Chinese black sleeper(Bostrychus sinensis)is a fish that lives both in seawater and freshwater,feeds on crustaceans,aquatic insects and occasionally shellfish.The existence of digestive enzyme in viscera to act on chi...Chinese black sleeper(Bostrychus sinensis)is a fish that lives both in seawater and freshwater,feeds on crustaceans,aquatic insects and occasionally shellfish.The existence of digestive enzyme in viscera to act on chitinous exoskeleton of the prey is of interest.In this study,a chitinase was purified to homogeneity using ammonium sulfate precipitation,DEAE-Sephacel ion exchange,Sephacryl S-200 HR and Superdex 200 gel filtration columns.The purified protein presents a molecular mass of 58 kDa as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE)and results in a single band on native PAGE.According to peptide mass fingerprinting,two peptides containing a total of 20 amino acid residues,were 95%identical to a chitinase from yellow perch(Perca flavescens)and 100%identical to the chitinase from greater amberjack(Seriola dumerili).The purified chitinase showed optimum activity at pH 6.0,and was stable at acidic conditions and temperature below 55℃.The enzymatic activity was quite stable in the presence of NaCl,even at 1 mol/L.The chitinase was capable of degrading chitosan into low molecular mass chitooligosaccharides(COS)with sizes in a range of 200-700 Da,and the circular dichroism profile of the COS greatly differed from native chitosan.Full-length cDNA encoding the present chitinase was cloned and the transcript levels of chitinase in various tissues were determined by quantitative real-time PCR.The results showed that the transcript level of chitinase was highest in esophagus and hepatopancreas.展开更多
Pre-harvest sprouting (PHS) reduces yields and grain quality, resulting in seriously economic losses in wheat. It has been showed that PHS is significantly correlated to seed dormancy levels. <em>FUSCA3</em&g...Pre-harvest sprouting (PHS) reduces yields and grain quality, resulting in seriously economic losses in wheat. It has been showed that PHS is significantly correlated to seed dormancy levels. <em>FUSCA3</em> (<em>FUS3</em>) gene is considered to be the key regulator of seed dormancy. However, little information is available about the function of <em>FUS3</em> gene (<em>TaFUS3</em>) in wheat. In this study, three homologous genes were identified in wheat grain, and their functions were investigated by gene silencing. Three full-length DNA (3477, 3534 and 3501 bp) and cDNA (1015, 1012 and 1015 bp) sequences encoding a B3 transcription factor, designated <em>TaFUS3-3A</em>, <em>TaFUS3-3B</em> and <em>TaFUS3-3D</em>, were first isolated from common wheat. The transcription of three <em>TaFUS3</em> genes in seed development and germination process was detected.<em> TaFUS3-3B</em> and<em> TaFUS3-3D</em> had similar expression profiles, and high levels of gene transcripts were detected in seeds at 25 DAP (days after pollination) and after 24 h of imbibition. However, the transcription of <em>TaFUS3-3A </em>was not detected. Silencing of <em>TaFUS3</em> in common wheat spikes resulted in increased seed germination and PHS. Compared with wild-type, the <em>TaFUS3</em>-silenced plants showed increased expression of genes related to GA biosynthesis and ABA metabolism, and decreased expression of genes associated with ABA biosynthesis. Moreover, silencing of <em>TaFUS3</em> in wheat plants led to a decrease in embryo sensitivity to ABA and changed the expression of genes involved in ABA signal transduction. The results of gene silencing indicated that<em> TaFUS3</em> plays a positive role in wheat seed dormancy and PHS-resistance, which might be associated with ABA, GA level and signal transduction.展开更多
The fatty acid elongase 1 (FAE1) genes of Brassic napus were cloned from two cultivars, i.e. Zhong- shuan No. 9 with low erucic acid content, and Zhongyou 821 with high erucic acid content, using the degenerate PCR pr...The fatty acid elongase 1 (FAE1) genes of Brassic napus were cloned from two cultivars, i.e. Zhong- shuan No. 9 with low erucic acid content, and Zhongyou 821 with high erucic acid content, using the degenerate PCR primers. The sequence analysis showed that there was no intron within the FAE1 genes. The FAE1 genes from Zhongyou 821 contained a coding sequence of 1521 nucleotides, and those cloned from Zhongshuan No. 9 contained a 1517 bp coding sequence. Alignment of the FAE1 sequences from Brassica rapa, B. oleracea and B. napus detected 31 single nucleotide polymorphic sites (2.03%), which resulted in 7 amino-acid substitutions. Further analysis indicated that 19 SNPs were genome-specific, of which, 95% were synonymous mutations. The nucleotide substitution at po- sition 1217 in the FAE1 genes led to a specific site of restricted cleavage. An AvrII cleavage site was present only in the C genome genes and absent in the A genome FAE1 genes. Digestion profile of the FAE1 sequences from B. rapa, B. oleracea and B. napus produced with AvrII confirmed that the FAE1 genes of B. oleracea origin was recognized and digested, while that of B. rapa origin could not. The results indicated that by AvrII cleavage it was possible to distinguish B. rapa from B. oleracea and be- tween the A and C genome of B. napus. In addition, the FAE1 genes could be used as marker genes to detect the pollen flow of B. napus, thus providing an alternative method for risk assessment of gene flow.展开更多
Glycyrrhiza uralensis is considered to be one of the most important herbs in traditional Chinese medicine due to its numerous pharmacological effects particularly its ability to relieve cough and act as a mucolytic.Ba...Glycyrrhiza uralensis is considered to be one of the most important herbs in traditional Chinese medicine due to its numerous pharmacological effects particularly its ability to relieve cough and act as a mucolytic.Based on previous research,these effects are mediated by a number of active ingredients,especially glycyrrhizic acid(GA).In the present study,a gene encodingβ-amyrin synthase(β-AS)involved in GA biosynthesis in G.uralensis has been cloned and expressed in Saccharomyces cerevisiae.The cloned enzyme showed similar activity to native enzymes isolated from other Glycyrrhiza species to catalyze the conversion of 2,3-oxidosqualene intoβ-amyrin.In fact theβ-AS gene is particularly important in the GA biosynthetic pathway in G.uralensis.The complete sequence of the enzyme was determined and a phylogenetic tree based on theβ-AS gene of G.uralensis and 20 other species was created.This showed that Glycyrrhiza glabra had the closest kinship with G.uralensis.The results of this work will be useful in determining how to improve the efficacy of G.uralensis by improving its GA content and in exploring the biosynthesis of GA in vitro.展开更多
Dehydration-responsive element-binding(DREB)proteins,specifically binding to the dehydration-responsive element(DRE),have been identified as a group of important transcription activators of plants which regulate the e...Dehydration-responsive element-binding(DREB)proteins,specifically binding to the dehydration-responsive element(DRE),have been identified as a group of important transcription activators of plants which regulate the expression of genes in response to drought,high-salt and lowtemperature stresses.Two DREB-like genes from Bermuda grass that are induced by low-temperature or high-salt stresses were cloned using RT-PCR and RACE methods,and were named BeDREB1 and BeDREB2,respectively(GenBank accession No:AY462117 and AY462118).They contained an ORF of 753 bp encoding 251 amino acids,showing the typical characteristics of the DREB gene family.Interestingly,these two genes isolated from Bermuda grass induced either by low-temperature stress or high-salt stress shared 97.8%homology.Furthermore,it was demonstrated that both BeDREB1 and BeDREB2 could bind to the wild-type DRE element to activate the transcription of the reporter gene HIS3,driven by a promoter carrying DRE cis-element in yeast strain 4721,in the presence of 3-AT.RT-PCR showed that BeDREB1 and BeDREB2 genes could be greatly induced by low-temperature and high-salt stresses,respectively.Their expressions were changed following the inducible time.In conclusion,all results indicate that BeDREB1 and BeDREB2 genes isolated from treated Bermuda grass are new members of the DREB transcription activator family,which may play very important roles in signal transduction related to stresses.展开更多
文摘The critical antioxidant catalase(CAT)breaks down hydrogen peroxide induced by environmental stresses.Here we cloned full length catalase cDNA from Lymantria dispar asiatic(LdCAT).Bioinformatic analyses showed that open reading frames of LdCAT contains 1524 bp,encoding 507 amino acids with molecular weight of 126.99 kDa,theoretical pI of 5.00,aliphatic index of 29.92,grand average of hydropathicity of 0.764,and instability index(II)of 46.56.Protein BLAST and multiple sequence alignment indicated that LdCAT had high identity with CAT from other insects,especially lepidopterans.In a phylogenetic analysis,LdCAT was most similar to CAT from Spodoptera litura and S.exigua.Quantitative realtime polymerase chain reaction showed that LdCAT transcripts in all instar larvae and the five tissues tested,verifying the ubiquity of LdCAT in L.disapr.Moreover,LdCAT of third instar larvae was significantly upregulated after they fed on avermectin at sublethal and LC10 doses.The highest relative transcript levels were found 2 h after an avermectin spray at LC90,and in the cuticula,rather than heads,fat bodies,malpighian tubes,and midguts after a spray avermectin at a sublethal concentration.The expression level of LdCAT under pesticide stresses here suggested that CAT is an important antioxidant enzyme of L.disapr defensing against pesticide stress and may be a good target for controlling this pest.
基金The National Basic Research Program of China(973 Program)under contract No.2013CB835304the National Marine Public Projects under contract No.201305016+2 种基金the National Natural Science Foundation of China(General Program)under contract No.31601865the Dalian Science and Technology Program under contract No.2013E11SF056the Education Department of the General Scientific Research Project under contract No.L201683651
文摘The Guanine nucleotide exchange factor Vav2(Vav2) is a member of the Vav family that serves as an important regulators for the Rho family of Ras-related GTPases. In the current study, an ortholog(Lj-Vav2) of Vav2 was identified in the lamprey(Lampetra japonica). To elucidate the phylogenetic relationship of Vav2, the metazoan genome databases were analyzed to mine the ortholog of Vav. It was found that Vav2 genes were only existed in vertebrates and Lj-Vav2 was the original one found in agnathans. The evolutionary dynamics of conserved motifs of Vav2 were explored using combined amino acid sequence as markers, and it is revealed that the Calponin homology(CH) domain, Dbl-homologous(DH) domain, Pleckstrin homology(PH) domain, Cysteine-rich(C1)domains, Src homology 3(SH3) domains and Src homology 2(SH2) domain were conserved throughout the Vav2 gene family in vertebrates during gene evolution. Relative quantitative real-time PCR analysis showed that the LjVav2 was distributed in the heart, kidney, supraneural myeloid body, liver, gill and lymphocyte-like cells. The LjVav2 was found to be expressed in these tissues, and the level of which was upregulated in lymphocyte-like cells after the animal was stimulated with LPS. These results indicated that the Lj-Vav2 might be involved in the immune response of lymphocyte-like cells in lamprey. Meanwhile, our findings provided a foundation for further investigation of the function of Lj-Vav2 in the primary vertebrate.
文摘Sea Island cotton(Gossypium barbadense L.) has been highly valued in Verticillium wilt resistance and many fiber qualities including fiber length,strength,and fineness.To identify whether
文摘Verticillium dahliae Kleb.is a necrotrophic plant pathogen which causes serious soil borne vascular disease in cotton.The molecular basis the defense response of cotton to this pathogen is
文摘CAP,an adenylyl cyclase-associated protein,is predicted to be involved in cytoskeletal organization and signal transduction.Recently,we found that CAP may play an important role in fuzz-like fiber cell initiation in cotton.For the further research,we isolated two CAP homologues from wild
文摘The zinc finger proteins belong to the largest family of transcription factors.But there is little research of Cys2/His2 type zinc finger proteins in cotton,and there is no submission of correlating
文摘Studies on the cold-responsive genes and cold signaling of woody species drop far behind in comparison to herbaceous plants.Due to similar lignified structure,perennial characteristic,and enhanced tolerance,it seems much easier to find strongly antifreeze genes and obtain effective results in transgenic woody plants.In this study,Ammopiptanthus mongolicus,an evergreen,broadleaf and cold-resist leguminous shrub growing in the desert of Inner Mongolia,was used as a material for low-temperature induced gene isolation.Through differential expression analysis induced by low-temperature,thirteen up-regulated cDNAs were identified.One of them,AmEBP1,(accession number:DQ519359)confers enhanced cold-tolerance to both transgenic E.coli and transgenic Arabidopsis.Results suggest that AmEBP1 can stimulate the synthesis of ribosome and the dephosphyration of the α-subunit of initiation factor 2(eIF2α),and subsequently promote the translation process.By which the transgenic plants obtained increased cold-resistant ability.
文摘In this study, we report that we successfully cloned and sequenced a chitinase gene from the ovotestis of Kuroda’s sea hare Aplysia kurodai. By using reverse transcription-polymerase chain reaction (RT-PCR) and a system for the 5’ and 3’ rapid amplification of cDNA ends, we obtained a 1352 bp chitinase gene (AkChi) from the ovotestis of A. kurodai. AkChi contains a 1263 bp open reading frame that encodes 421 amino acids. The domain structure predicted from the deduced amino acid sequence was an N-terminal signal peptide and a catalytic domain of glycoside hydrolase (GH) family 18 chitinase. A comparative analysis of the deduced amino acid sequences of AkChi with those of the acidic mammalian chitinase of the California sea hare Aplysia californica revealed the highest homology at 83%. The purified chitinase from the ovotestis was digested by trypsin, and 119 residues of digested peptides were consistent with the deduced amino acid sequence of AkChi. We used RT-PCR to evaluate the expression of AkChi in various tissues of A. kurodai, and we observed that AkChi was expressed only in the ovotestis. A phylogenetic tree analysis, performed using the amino acid sequences of AkChi and known GH family 18 chitinases, showed that AkChi was separated from the molluscan chitinases with a chitin binding domain. To our knowledge, this is the first study demonstrating the cDNA cloning of an ovotestis chitinase from a sea hare.
基金supported by the National Key R&D Program of China(No.2017YFC1701200).
文摘Phenylalanine ammonia-lyase(PAL),which catalyzes the conversion from L-phenylalanine to trans-cinnamic acid,is a well-known key enzyme and a connecting step between primary and secondary metabolisms in the phenylpropanoid biosynthetic pathway of plants and microbes.Schisandra chinensis,a woody vine plant belonging to the family of Magnoliaceae,is a rich source of dibenzocyclooctadiene lignans exhibiting potent activity.However,the functional role of PAL in the biosynthesis of lignan is relatively limited,compared with those in lignin and flavonoids biosynthesis.Therefore,it is essential to clone and characterize the PAL genes from this valuable medicinal plant.In this study,molecular cloning and characterization of three PAL genes(ScPAL1−3)from S.chinensis was carried out.ScPALs were cloned using RACE PCR.The sequence analysis of the three ScPALs was carried out to give basic characteristics followed by docking analysis.In order to determine their catalytic activity,recombinant protein was obtained by heterologous expression in pCold-TF vector in Escherichia coli(BL21-DE3),followed by Ni-affinity purification.The catalytic product of the purified recombinant proteins was verified using RP-HPLC through comparing with standard compounds.The optimal temperature,pH value and effects of different metal ions were determined.Vmax,Kcat and Km values were determined under the optimal conditions.The expression of three ScPALs in different tissues was also determined.Our work provided essential information for the function of ScPALs.
基金supported by the National Science Foundation for Distinguished Young Scholars of China(Grant No.31325024)Innovation Team Project of Shandong Modern Agricultural Industry Technology System(SDAIT-03-022-03)
文摘A hexokinase gene named MdHXK1(MDP0000309677) was cloned from ‘Gala' apple(Malus × domestica Borkh.). Sequence analysis showed that the MdHXK1 gene was 1 497 bp long and encoded 499 amino acids. The predicted molecular mass of this protein was 54.05 kD, and the pI was 5.76. A phylogenetic tree indicated apple MdHXK1 exhibited the highest sequence similarity to Pyrus bretschneideri PbHXK1. Analysis of the functional domain showed that the MdHXK1 protein included two conserved kinase domains. The prediction of subcellular localization suggested that the MdHXK1 protein was mainly localized in the cytoplasm. There was an indication that MdHXK1 existed as one copy in the apple genome by Southern blotting. Silico analysis suggested that the promoter sequence contained several typical cis-acting elements, including defense, sugar signaling and phytohormone responsive elements. Quantitative real-time PCR analysis demonstrated that the MdHXK1 gene was mainly expressed in stem and flower tissues. During the development of apple fruits, the expression of the MdHXK1 gene initially increased and then decreased. The changes on Glc phosphorylation relative activity and glucose concentration showed the same trend. In addition, the expression of this gene was induced by salt stress, low temperature, and abscisic acid(ABA). Finally, we obtained and purified the fused MdHXK1 protein by recombinant prokaryotic expression. Studies have demonstrated that MdHXK1 may participate in sugar metabolism in apple fruits. Enzyme encoded by MdHXK1 is a key factor in the mediation of sugar accumulation. Recently,researchers on hexokinase at home and abroad mainly focused on model plants, such as Arabidopsis, tobacco and rice, but orchard fruit like apple were underresearched. Our research established the foundation for the further study of the functions of MdHXK1.
基金The National Natural Science Foundation Committee(No. 39770823)
文摘Objective To identlfy the member of the caspase famlly proteases involved in Y-radiation-inducedapoptosis in HL-60 cells and to study the expression or the caspase gene in normal, apoptotic cells and in immortal tumor cells. Methods By using degenerate oligonucleotide primers encoding the highly conserved peptides that were present in all known caspases, we performed RT-PCR on poly(A)RNA from γ-radiation-induced apoptotic HL-6o cells.Caspase-3 mRNA in apoptotic HL-6o cells and in human tumor cell lines was analyzed by Northern blot. Results The amplified DNA fragment was identiried with caspase-3 cDNA by cloning and sequencing. The Northern blot analysis of caspase-3 mRNA of different human tumor cell lines showed that the caspase-3 gene transcript was more highly expressed in leukemia cell lines and the SH-SY5Y cell line than in HeLa and MCF-7 cells. It was more highly expressed in the radiation-induced apoptotic HL-6o cells than in control HL-6o cells. Conclusion These results indicated that caspase-3 was involved in γ-radiation-induced apoptosis in HL-6o cells. The high level of expression or caspase-3 may aid efforts to understand the insensitivity of some tumor cells to radlation, their inherent ability to survive, and
基金The National Key R&D Program of China under contract No.2018YFD0901004the National Natural Science Foundation of China under contract Nos 31772049,31702372。
文摘Chinese black sleeper(Bostrychus sinensis)is a fish that lives both in seawater and freshwater,feeds on crustaceans,aquatic insects and occasionally shellfish.The existence of digestive enzyme in viscera to act on chitinous exoskeleton of the prey is of interest.In this study,a chitinase was purified to homogeneity using ammonium sulfate precipitation,DEAE-Sephacel ion exchange,Sephacryl S-200 HR and Superdex 200 gel filtration columns.The purified protein presents a molecular mass of 58 kDa as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE)and results in a single band on native PAGE.According to peptide mass fingerprinting,two peptides containing a total of 20 amino acid residues,were 95%identical to a chitinase from yellow perch(Perca flavescens)and 100%identical to the chitinase from greater amberjack(Seriola dumerili).The purified chitinase showed optimum activity at pH 6.0,and was stable at acidic conditions and temperature below 55℃.The enzymatic activity was quite stable in the presence of NaCl,even at 1 mol/L.The chitinase was capable of degrading chitosan into low molecular mass chitooligosaccharides(COS)with sizes in a range of 200-700 Da,and the circular dichroism profile of the COS greatly differed from native chitosan.Full-length cDNA encoding the present chitinase was cloned and the transcript levels of chitinase in various tissues were determined by quantitative real-time PCR.The results showed that the transcript level of chitinase was highest in esophagus and hepatopancreas.
文摘Pre-harvest sprouting (PHS) reduces yields and grain quality, resulting in seriously economic losses in wheat. It has been showed that PHS is significantly correlated to seed dormancy levels. <em>FUSCA3</em> (<em>FUS3</em>) gene is considered to be the key regulator of seed dormancy. However, little information is available about the function of <em>FUS3</em> gene (<em>TaFUS3</em>) in wheat. In this study, three homologous genes were identified in wheat grain, and their functions were investigated by gene silencing. Three full-length DNA (3477, 3534 and 3501 bp) and cDNA (1015, 1012 and 1015 bp) sequences encoding a B3 transcription factor, designated <em>TaFUS3-3A</em>, <em>TaFUS3-3B</em> and <em>TaFUS3-3D</em>, were first isolated from common wheat. The transcription of three <em>TaFUS3</em> genes in seed development and germination process was detected.<em> TaFUS3-3B</em> and<em> TaFUS3-3D</em> had similar expression profiles, and high levels of gene transcripts were detected in seeds at 25 DAP (days after pollination) and after 24 h of imbibition. However, the transcription of <em>TaFUS3-3A </em>was not detected. Silencing of <em>TaFUS3</em> in common wheat spikes resulted in increased seed germination and PHS. Compared with wild-type, the <em>TaFUS3</em>-silenced plants showed increased expression of genes related to GA biosynthesis and ABA metabolism, and decreased expression of genes associated with ABA biosynthesis. Moreover, silencing of <em>TaFUS3</em> in wheat plants led to a decrease in embryo sensitivity to ABA and changed the expression of genes involved in ABA signal transduction. The results of gene silencing indicated that<em> TaFUS3</em> plays a positive role in wheat seed dormancy and PHS-resistance, which might be associated with ABA, GA level and signal transduction.
基金the National Natural Science Foundation of China (Grant No. 30471099)Development Plan of the State Key Fundamental Research of China (Grant No. 2006CB101600)the National High Technology and Development Program of China (Grant No. 2006AA10A113)
文摘The fatty acid elongase 1 (FAE1) genes of Brassic napus were cloned from two cultivars, i.e. Zhong- shuan No. 9 with low erucic acid content, and Zhongyou 821 with high erucic acid content, using the degenerate PCR primers. The sequence analysis showed that there was no intron within the FAE1 genes. The FAE1 genes from Zhongyou 821 contained a coding sequence of 1521 nucleotides, and those cloned from Zhongshuan No. 9 contained a 1517 bp coding sequence. Alignment of the FAE1 sequences from Brassica rapa, B. oleracea and B. napus detected 31 single nucleotide polymorphic sites (2.03%), which resulted in 7 amino-acid substitutions. Further analysis indicated that 19 SNPs were genome-specific, of which, 95% were synonymous mutations. The nucleotide substitution at po- sition 1217 in the FAE1 genes led to a specific site of restricted cleavage. An AvrII cleavage site was present only in the C genome genes and absent in the A genome FAE1 genes. Digestion profile of the FAE1 sequences from B. rapa, B. oleracea and B. napus produced with AvrII confirmed that the FAE1 genes of B. oleracea origin was recognized and digested, while that of B. rapa origin could not. The results indicated that by AvrII cleavage it was possible to distinguish B. rapa from B. oleracea and be- tween the A and C genome of B. napus. In addition, the FAE1 genes could be used as marker genes to detect the pollen flow of B. napus, thus providing an alternative method for risk assessment of gene flow.
文摘Glycyrrhiza uralensis is considered to be one of the most important herbs in traditional Chinese medicine due to its numerous pharmacological effects particularly its ability to relieve cough and act as a mucolytic.Based on previous research,these effects are mediated by a number of active ingredients,especially glycyrrhizic acid(GA).In the present study,a gene encodingβ-amyrin synthase(β-AS)involved in GA biosynthesis in G.uralensis has been cloned and expressed in Saccharomyces cerevisiae.The cloned enzyme showed similar activity to native enzymes isolated from other Glycyrrhiza species to catalyze the conversion of 2,3-oxidosqualene intoβ-amyrin.In fact theβ-AS gene is particularly important in the GA biosynthetic pathway in G.uralensis.The complete sequence of the enzyme was determined and a phylogenetic tree based on theβ-AS gene of G.uralensis and 20 other species was created.This showed that Glycyrrhiza glabra had the closest kinship with G.uralensis.The results of this work will be useful in determining how to improve the efficacy of G.uralensis by improving its GA content and in exploring the biosynthesis of GA in vitro.
基金This paper was supported by the National High Technology Research and Development Program of China(863 Pro-gram)(No.2002AA224091).
文摘Dehydration-responsive element-binding(DREB)proteins,specifically binding to the dehydration-responsive element(DRE),have been identified as a group of important transcription activators of plants which regulate the expression of genes in response to drought,high-salt and lowtemperature stresses.Two DREB-like genes from Bermuda grass that are induced by low-temperature or high-salt stresses were cloned using RT-PCR and RACE methods,and were named BeDREB1 and BeDREB2,respectively(GenBank accession No:AY462117 and AY462118).They contained an ORF of 753 bp encoding 251 amino acids,showing the typical characteristics of the DREB gene family.Interestingly,these two genes isolated from Bermuda grass induced either by low-temperature stress or high-salt stress shared 97.8%homology.Furthermore,it was demonstrated that both BeDREB1 and BeDREB2 could bind to the wild-type DRE element to activate the transcription of the reporter gene HIS3,driven by a promoter carrying DRE cis-element in yeast strain 4721,in the presence of 3-AT.RT-PCR showed that BeDREB1 and BeDREB2 genes could be greatly induced by low-temperature and high-salt stresses,respectively.Their expressions were changed following the inducible time.In conclusion,all results indicate that BeDREB1 and BeDREB2 genes isolated from treated Bermuda grass are new members of the DREB transcription activator family,which may play very important roles in signal transduction related to stresses.
