TDF1(transcription-drived fragment) was homologous to the predicted S. lycopersicum nonspecific lipid-transfer protein,nsLTP 2-like(91%), and it was significantly upregulated in response to C. fulvum(cladosporium fulv...TDF1(transcription-drived fragment) was homologous to the predicted S. lycopersicum nonspecific lipid-transfer protein,nsLTP 2-like(91%), and it was significantly upregulated in response to C. fulvum(cladosporium fulvum) infection in tomato plants.In this experiment, the full-length cDNA of nsLTP 2-like was cloned using RACE technology based on the sequence of TDF1(GenBank: JZ717725). A full-length, 625 bp(GenBank: KU366289), cDNA sequence, which with 98% similarity to nsLTP 2-like gene(GenBank: XM015233692) was obtained. This cDNA contains an ORF(open reading frame) with full-length of 345 bp, coding of 114 amino acids, including 12.3% Ala and Gly. Protein molecular weight was 11.51 ku, the isoelectric point(pI) was 8.99, and average overall hydrophilicity was 0.412, with one phosphorylation sites, belonging to volatile acidic nuclear protein. Secondary structure prediction showed that α-Helix accounts for 30.7%, extension chain for 12.28%, β-corner for 9.65%, and random coil for 47.37%. Through comparative analysis of the homology among species, it was found that the amino acid sequence of tomato nsLTP 2-like protein had a high similarity with other plants, and with a specific conserved sequence which might related features in nsLTP 2-like protein. It also be analyzed the gene expression pattern of tomato in different parts and under different stress conditions.The results showed that nsLTP 2-like gene was up-regulated in varying degrees, under the condition of cold stress, exogenous hormone spraying and cladosporium fulvum infection. Therefore, it was speculated that the gene played a role in response to abiotic and biotic stress in tomato.展开更多
[Objective] The aim of this study is to clone bovine interleukin-2 gene(IL-2)and observe its expression in prokaryotic cells.[Method]Bovine IL-2 gene was amplified from total RNA of peripheral blood lymphocytes of hol...[Objective] The aim of this study is to clone bovine interleukin-2 gene(IL-2)and observe its expression in prokaryotic cells.[Method]Bovine IL-2 gene was amplified from total RNA of peripheral blood lymphocytes of holstein-friesian cows by RT-PCR.Subsequently,the gene was cloned into pGEX-2T prokaryotic expression plasmid to construct recombinant,which was then transformed into Escherichia Coli BL21.After IPTG induction,SDS-PAGE analysis was conducted.[Result]A 500 bp target fragment corresponding with expectation was obtained by RT-PCR.The cloned gene successfully expressed fusion protein of about 43 kD in prokaryotic cells.[Conclusion]This study provided a theoretical and material basis for further researches on IL-2 gene.展开更多
In this study,to clarify the bioactive polypeptides included in the skins and secretions of Bufo,we screened the Japanese toad(Bufo japonicus formosus) skin cDNA library by colony polymerase chain reaction(PCR),and ob...In this study,to clarify the bioactive polypeptides included in the skins and secretions of Bufo,we screened the Japanese toad(Bufo japonicus formosus) skin cDNA library by colony polymerase chain reaction(PCR),and obtained a transcript of 1 075 bp consisting of 1 37 bp 5′ untranslated region(UTR),515 bp 3′ UTR and a 423 bp open reading frame(ORF) encoding a polypeptide of 140 amino acid residues(GenBank accession number: KF359945).Homolog analysis showed a 70%–96% homology with sterol carrier protein-2(SCP-2) present in other animals,which is implicated in lipid metabolism of other organisms.The gene SCP-2 of Chinese toad(B.gargarizans) was cloned from a first strand cDNA of Bufo skin(GenBank accession number: KF381341) via PCR,whose encoding polypeptide has only one amino acid difference from that of Japanese toad.Tissue distribution analysis showed that SCP-2 expressed in all organs tested,though in the liver and spleen it manifested lower expression than in other organs.These findings might indicate SCP-2 being one of the active ingredients in toad skin.These findings may in turn have implications for further drug development from traditional Chinese medicine sources.展开更多
At present, the research about flower color of Rosa rugosa is a very inno-vative and practical study. Glycosylation modification fulfills an important role in increasing the stability and solubility of anthocyanin in ...At present, the research about flower color of Rosa rugosa is a very inno-vative and practical study. Glycosylation modification fulfills an important role in increasing the stability and solubility of anthocyanin in plants. In this study, based on the transcriptional database of R. rugosa, a gene with full length cDNA of 1422bp, encoding 473 amino acids, designated as RrGT2, were isolated from flowers of R. rugosa ‘Zizhi’ and then functionally characterized. According to online software prediction, the molecular formula of the protein encoded by the RrGT2 gene is C2334H3628N602O711S18, the relative molecular mass is 52,075.17 Da, and the theoretical isoelectric point is pI = 4.76. The result of the RrGT2 protein 3D model construction showed that it had the highest homology with the UDP-glycosyltransferase 74F2 protein model in the database (39.53%). Sequence alignments with the NCBI database showed that the RrGT2 protein is a member of the GTB superfamily. Homology analysis revealed that the coding regions of RrGT2 was highly specific among different species, but still had typical conserved amino acid residues called PSPG that are crucial for RrGT2 enzyme activity. RrGT2 transcripts were detected in five flowering stages and seven tissues of R. rugosa ‘Zizhi’, R. rugosa ‘Fenzizhi’ and R. rugosa ‘Baizizhi’, and their expression patterns corresponded with the accumulation of antho-cyanins. Therefore, we speculated that glycosylation of RrGT2 plays a crucial role in anthocyanin biosynthesis in R. rugosa.展开更多
[ Objective] To clone and express canine IL-2 gene and thus to provide theoretical support for the development of novel immune enhancers and genetic engineering vaccines. [ Method] Leukocytes separated from canine who...[ Objective] To clone and express canine IL-2 gene and thus to provide theoretical support for the development of novel immune enhancers and genetic engineering vaccines. [ Method] Leukocytes separated from canine whole blood were stimulated by concanavalin for 20 h, and then total RNA was extracted. According to the sequence of canine IL-2 gene published in the GenBank, a pair of primers was designed. After PCR am- plificetion, the target fragment was cloned into prokaryotic expression vector pET-28a. The recombinants were transformed into the host bacteria BL21. After IPTG induction, the expression products were analyzed by SDS-PAGE. [ Result] A 500 bp band with the expected size appeared in the RT-PCR products. After the pMD18-T-IL2 was identified by double digestion, an approximately 500 bp fragment was produced, which indicated successful cloning of the gene. After the pET-28a-lL2 was identified by restriction enzyme digestion and PCR, a 500 bp fragment was produced, which indicated successful construction of the expression vector. As revealed by the SDS-PAGE analysis, a protein band with molecular weight of about 20 kDa appeared. [ Conclusion] The canine IL-2 gene was cloned and expressed.展开更多
A repeated sequence DNA fragment, L5B-4, was cloned from the 5 kb BamHI DNA fragments of rat genomic DNA. The expressions of the L5B-4 DNA fragment are different in liver and hepatoma cells. The amounts of transcripts...A repeated sequence DNA fragment, L5B-4, was cloned from the 5 kb BamHI DNA fragments of rat genomic DNA. The expressions of the L5B-4 DNA fragment are different in liver and hepatoma cells. The amounts of transcripts in hepatoma cells are lower in nucleus and higher in cytoplasm, especially in polysomal RNA, as compared with that in liver cells. The alteration shown in polysomal RNA of hepatoma cells seems to be specific. These results are discussed with respect to the possible function of this repeated DNA and its variation in hepatoma cells.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
Both copper/zinc superoxide dismutase （SOD; Cu/Zn-SOD, SOD1） cDNA and manganese SOD （Mn-SOD, SOD2） cDNA were cloned for the first time from the three-keeled pond turtle, Chinemys reevesii, using RT-PCR and RACE me...Both copper/zinc superoxide dismutase （SOD; Cu/Zn-SOD, SOD1） cDNA and manganese SOD （Mn-SOD, SOD2） cDNA were cloned for the first time from the three-keeled pond turtle, Chinemys reevesii, using RT-PCR and RACE methods in this work. The SOD1 cDNA was 749 bp long and consisted of a 32-bp 5＇-untranslated region （UTR）, a 249-bp 3＇-UTR, and a 468-bp open reading frame （ORF） encoding a 155-amino-acid protein with 16.0 kDa predicted molecular mass and 5.95 theoretical isoelectric point （p/）. The SOD2 cDNA was 1687 bp long and comprised 94-bp of 5＇-UTR, 912-bp 3＇-UTR and 681-bp ORF encoding a 226-amino-acid protein with 25.0 kDa predicted molecular mass and 8.83 pI. The deduced amino acid sequence of SOD1 showed relatively high similarity （77.4%-87.1%） and identity （65.4%-74.4%） with the published sequences of SOD1 from other vertebrate species, whereas SOD2 protein shared slightly higher similarity （83.6%-95.6%） and identity （76.1%-88.9%） with other reported vertebrates SOD2s. Phylogenetic analysis revealed that the C. reevesii SOD1 and SOD2 were separately clustered together, and were highly conserved during evolution. Both SOD mRNA expression was detected widely in the brain, liver, muscle, kidney, gut, spleen, lung and heart at variable levels. The highest expression of the two SODs was observed in muscle, and followed in brain, liver, kidney, gut and heart, whereas low transcriptional levels were found in spleen and lung. Meanwhile, high activity of SOD 1 was kept in brain, liver, muscle, kidney and heart, and followed in gut, spleen and lung. The activities of SOD2 in brain, liver, muscle, kidney, gut and heart were significantly higher than those in spleen and lung.展开更多
To determine the pathogenic potential of the leptospiral flagella-associated proteins, the genes flhA and flhB2 encoding the biosynthesis of flagella of leptospira interrogans serogroup icterohaemor- rhagiae serovar l...To determine the pathogenic potential of the leptospiral flagella-associated proteins, the genes flhA and flhB2 encoding the biosynthesis of flagella of leptospira interrogans serogroup icterohaemor- rhagiae serovar lai stain 56601 were cloned and their prokaryotic expression systems were constructed. It was demonstrated that the cloned flhA and flhB2 genes had 2118 bp in length and showed 100% and 99.9% of homologies in their nucleotide sequences and 100% and 98.8% of homologies in their putative amino acid sequences respectively, in comparison with those of previously reported. The prokaryotic expression systems under the induction with IPTG could efficiently express the target proteins rFlhA and rFlhB2 with the outputs of approximate 10% of the total bacterial proteins. Based on the sequences of the cloned genes flhA and flhB2, the structural features in associated with pathogenesis and the functions of the target proteins were analyzed with bioinformatics softwares, in which the FlhA was found to have 7 major transmembrane helices, while the FlhB2 had 5 ones. The conserved domains in the FlhA showed high similarity to those of the FHIPEP of the other bacterial FlhA and EscV families, but the conserved domains in the FlhB2 were similar to those of bac-export-2 and EscU families, EscV and EscU families being the protein products of the type IR secretion system in association with pathogenesis. The FlhA and FlhB2 also contained protein kinase C （PKC） and protein tyrosine kinase （PTK） phosphorylation sites, indicating that PKC and PTK of host cells were involved in the internalization and intracellular proliferation in the pathogenesis of microorganisms. All these data leads to a conclusion that the flhA and flhB2 genes of L. interrogans are relatively conserved and their gene products have great potential in the pathogenesis of this organism.展开更多
基金Supported by the National Key R&D Program of China(2017YFD0101900)China Agriculture Research System(CARS-23-A-16)the Science Foundation of Heilongjiang Province(C2017024)
文摘TDF1(transcription-drived fragment) was homologous to the predicted S. lycopersicum nonspecific lipid-transfer protein,nsLTP 2-like(91%), and it was significantly upregulated in response to C. fulvum(cladosporium fulvum) infection in tomato plants.In this experiment, the full-length cDNA of nsLTP 2-like was cloned using RACE technology based on the sequence of TDF1(GenBank: JZ717725). A full-length, 625 bp(GenBank: KU366289), cDNA sequence, which with 98% similarity to nsLTP 2-like gene(GenBank: XM015233692) was obtained. This cDNA contains an ORF(open reading frame) with full-length of 345 bp, coding of 114 amino acids, including 12.3% Ala and Gly. Protein molecular weight was 11.51 ku, the isoelectric point(pI) was 8.99, and average overall hydrophilicity was 0.412, with one phosphorylation sites, belonging to volatile acidic nuclear protein. Secondary structure prediction showed that α-Helix accounts for 30.7%, extension chain for 12.28%, β-corner for 9.65%, and random coil for 47.37%. Through comparative analysis of the homology among species, it was found that the amino acid sequence of tomato nsLTP 2-like protein had a high similarity with other plants, and with a specific conserved sequence which might related features in nsLTP 2-like protein. It also be analyzed the gene expression pattern of tomato in different parts and under different stress conditions.The results showed that nsLTP 2-like gene was up-regulated in varying degrees, under the condition of cold stress, exogenous hormone spraying and cladosporium fulvum infection. Therefore, it was speculated that the gene played a role in response to abiotic and biotic stress in tomato.
文摘[Objective] The aim of this study is to clone bovine interleukin-2 gene(IL-2)and observe its expression in prokaryotic cells.[Method]Bovine IL-2 gene was amplified from total RNA of peripheral blood lymphocytes of holstein-friesian cows by RT-PCR.Subsequently,the gene was cloned into pGEX-2T prokaryotic expression plasmid to construct recombinant,which was then transformed into Escherichia Coli BL21.After IPTG induction,SDS-PAGE analysis was conducted.[Result]A 500 bp target fragment corresponding with expectation was obtained by RT-PCR.The cloned gene successfully expressed fusion protein of about 43 kD in prokaryotic cells.[Conclusion]This study provided a theoretical and material basis for further researches on IL-2 gene.
基金Foundation items： This study was supported by the National Natural Science Foundation of China （31071181, 31372149） and the Students＇ Innovative Training Program of ZAFU （20120207, 20120213）
文摘In this study,to clarify the bioactive polypeptides included in the skins and secretions of Bufo,we screened the Japanese toad(Bufo japonicus formosus) skin cDNA library by colony polymerase chain reaction(PCR),and obtained a transcript of 1 075 bp consisting of 1 37 bp 5′ untranslated region(UTR),515 bp 3′ UTR and a 423 bp open reading frame(ORF) encoding a polypeptide of 140 amino acid residues(GenBank accession number: KF359945).Homolog analysis showed a 70%–96% homology with sterol carrier protein-2(SCP-2) present in other animals,which is implicated in lipid metabolism of other organisms.The gene SCP-2 of Chinese toad(B.gargarizans) was cloned from a first strand cDNA of Bufo skin(GenBank accession number: KF381341) via PCR,whose encoding polypeptide has only one amino acid difference from that of Japanese toad.Tissue distribution analysis showed that SCP-2 expressed in all organs tested,though in the liver and spleen it manifested lower expression than in other organs.These findings might indicate SCP-2 being one of the active ingredients in toad skin.These findings may in turn have implications for further drug development from traditional Chinese medicine sources.
文摘At present, the research about flower color of Rosa rugosa is a very inno-vative and practical study. Glycosylation modification fulfills an important role in increasing the stability and solubility of anthocyanin in plants. In this study, based on the transcriptional database of R. rugosa, a gene with full length cDNA of 1422bp, encoding 473 amino acids, designated as RrGT2, were isolated from flowers of R. rugosa ‘Zizhi’ and then functionally characterized. According to online software prediction, the molecular formula of the protein encoded by the RrGT2 gene is C2334H3628N602O711S18, the relative molecular mass is 52,075.17 Da, and the theoretical isoelectric point is pI = 4.76. The result of the RrGT2 protein 3D model construction showed that it had the highest homology with the UDP-glycosyltransferase 74F2 protein model in the database (39.53%). Sequence alignments with the NCBI database showed that the RrGT2 protein is a member of the GTB superfamily. Homology analysis revealed that the coding regions of RrGT2 was highly specific among different species, but still had typical conserved amino acid residues called PSPG that are crucial for RrGT2 enzyme activity. RrGT2 transcripts were detected in five flowering stages and seven tissues of R. rugosa ‘Zizhi’, R. rugosa ‘Fenzizhi’ and R. rugosa ‘Baizizhi’, and their expression patterns corresponded with the accumulation of antho-cyanins. Therefore, we speculated that glycosylation of RrGT2 plays a crucial role in anthocyanin biosynthesis in R. rugosa.
基金funded by the Project of Liaoning Education Department (L2010263)
文摘[ Objective] To clone and express canine IL-2 gene and thus to provide theoretical support for the development of novel immune enhancers and genetic engineering vaccines. [ Method] Leukocytes separated from canine whole blood were stimulated by concanavalin for 20 h, and then total RNA was extracted. According to the sequence of canine IL-2 gene published in the GenBank, a pair of primers was designed. After PCR am- plificetion, the target fragment was cloned into prokaryotic expression vector pET-28a. The recombinants were transformed into the host bacteria BL21. After IPTG induction, the expression products were analyzed by SDS-PAGE. [ Result] A 500 bp band with the expected size appeared in the RT-PCR products. After the pMD18-T-IL2 was identified by double digestion, an approximately 500 bp fragment was produced, which indicated successful cloning of the gene. After the pET-28a-lL2 was identified by restriction enzyme digestion and PCR, a 500 bp fragment was produced, which indicated successful construction of the expression vector. As revealed by the SDS-PAGE analysis, a protein band with molecular weight of about 20 kDa appeared. [ Conclusion] The canine IL-2 gene was cloned and expressed.
文摘A repeated sequence DNA fragment, L5B-4, was cloned from the 5 kb BamHI DNA fragments of rat genomic DNA. The expressions of the L5B-4 DNA fragment are different in liver and hepatoma cells. The amounts of transcripts in hepatoma cells are lower in nucleus and higher in cytoplasm, especially in polysomal RNA, as compared with that in liver cells. The alteration shown in polysomal RNA of hepatoma cells seems to be specific. These results are discussed with respect to the possible function of this repeated DNA and its variation in hepatoma cells.
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
基金supported by a grant from the National Natural Science Foundation of China (Project No. 31172383)
文摘Both copper/zinc superoxide dismutase （SOD; Cu/Zn-SOD, SOD1） cDNA and manganese SOD （Mn-SOD, SOD2） cDNA were cloned for the first time from the three-keeled pond turtle, Chinemys reevesii, using RT-PCR and RACE methods in this work. The SOD1 cDNA was 749 bp long and consisted of a 32-bp 5＇-untranslated region （UTR）, a 249-bp 3＇-UTR, and a 468-bp open reading frame （ORF） encoding a 155-amino-acid protein with 16.0 kDa predicted molecular mass and 5.95 theoretical isoelectric point （p/）. The SOD2 cDNA was 1687 bp long and comprised 94-bp of 5＇-UTR, 912-bp 3＇-UTR and 681-bp ORF encoding a 226-amino-acid protein with 25.0 kDa predicted molecular mass and 8.83 pI. The deduced amino acid sequence of SOD1 showed relatively high similarity （77.4%-87.1%） and identity （65.4%-74.4%） with the published sequences of SOD1 from other vertebrate species, whereas SOD2 protein shared slightly higher similarity （83.6%-95.6%） and identity （76.1%-88.9%） with other reported vertebrates SOD2s. Phylogenetic analysis revealed that the C. reevesii SOD1 and SOD2 were separately clustered together, and were highly conserved during evolution. Both SOD mRNA expression was detected widely in the brain, liver, muscle, kidney, gut, spleen, lung and heart at variable levels. The highest expression of the two SODs was observed in muscle, and followed in brain, liver, kidney, gut and heart, whereas low transcriptional levels were found in spleen and lung. Meanwhile, high activity of SOD 1 was kept in brain, liver, muscle, kidney and heart, and followed in gut, spleen and lung. The activities of SOD2 in brain, liver, muscle, kidney, gut and heart were significantly higher than those in spleen and lung.
文摘To determine the pathogenic potential of the leptospiral flagella-associated proteins, the genes flhA and flhB2 encoding the biosynthesis of flagella of leptospira interrogans serogroup icterohaemor- rhagiae serovar lai stain 56601 were cloned and their prokaryotic expression systems were constructed. It was demonstrated that the cloned flhA and flhB2 genes had 2118 bp in length and showed 100% and 99.9% of homologies in their nucleotide sequences and 100% and 98.8% of homologies in their putative amino acid sequences respectively, in comparison with those of previously reported. The prokaryotic expression systems under the induction with IPTG could efficiently express the target proteins rFlhA and rFlhB2 with the outputs of approximate 10% of the total bacterial proteins. Based on the sequences of the cloned genes flhA and flhB2, the structural features in associated with pathogenesis and the functions of the target proteins were analyzed with bioinformatics softwares, in which the FlhA was found to have 7 major transmembrane helices, while the FlhB2 had 5 ones. The conserved domains in the FlhA showed high similarity to those of the FHIPEP of the other bacterial FlhA and EscV families, but the conserved domains in the FlhB2 were similar to those of bac-export-2 and EscU families, EscV and EscU families being the protein products of the type IR secretion system in association with pathogenesis. The FlhA and FlhB2 also contained protein kinase C （PKC） and protein tyrosine kinase （PTK） phosphorylation sites, indicating that PKC and PTK of host cells were involved in the internalization and intracellular proliferation in the pathogenesis of microorganisms. All these data leads to a conclusion that the flhA and flhB2 genes of L. interrogans are relatively conserved and their gene products have great potential in the pathogenesis of this organism.
