Background:Tumor cell heterogeneity mediated drug resistance has been recognized as the stumbling block of cancer treatment.Elucidating the cytotoxicity of anticancer drugs at single-cell level in a high-throughput wa...Background:Tumor cell heterogeneity mediated drug resistance has been recognized as the stumbling block of cancer treatment.Elucidating the cytotoxicity of anticancer drugs at single-cell level in a high-throughput way is thus of great value for developing precision therapy.However,current techniques suffer from limitations in dynamically characterizing the responses of thousands of single cells or cell clones presented to multiple drug conditions.Methods:We developed a new microfluidics-based“SMART”platform that is Simple to operate,able to generate a Massive single-cell array and Multiplex drug concentrations,capable of keeping cells Alive,Retainable and Trackable in the microchambers.These features are achieved by integrating a Microfluidic chamber Array(4320 units)and a sixConcentration gradient generator(MAC),which enables highly efficient analysis of leukemia drug effects on single cells and cell clones in a high-throughput way.Results:A simple procedure produces 6 on-chip drug gradients to treat more than 3000 single cells or single-cell derived clones and thus allows an efficient and precise analysis of cell heterogeneity.The statistic results reveal that Imatinib(Ima)and Resveratrol(Res)combination treatment on single cells or clones is much more efficient than Ima or Res single drug treatment,indicated by the markedly reduced half maximal inhibitory concentration(IC50).Additionally,single-cell derived clones demonstrate a higher IC_(50) in each drug treatment compared to single cells.Moreover,primary cells isolated from two leukemia patients are also found with apparent heterogeneity upon drug treatment on MAC.Conclusions:This microfluidics-based“SMART”platform allows high-throughput single-cell capture and culture,dynamic drug-gradient treatment and cell response monitoring,which represents a new approach to efficiently investigate anticancer drug effects and should benefit drug discovery for leukemia and other cancers.展开更多
Potentilla sericea is a heavy metal hyperaccumulator landscaping plant.MYB transcription factors play an important role in regulating plant stress response to adversity.However,there are few studies on MYB transcripti...Potentilla sericea is a heavy metal hyperaccumulator landscaping plant.MYB transcription factors play an important role in regulating plant stress response to adversity.However,there are few studies on MYB transcription factors in stress tolerance in Potentilla sericea.In this study,the PsMYB62 gene was successfully cloned from Potentilla sericea.Methods:Bioinformatic analysis and real-time quantitative PCR(qPCR)methods were used to evaluate this gene.The transgenic A.thaliana were obtained by flower dipping and the gene function was identified by determining physiological indicators under cadmium stress.Results:The open reading frame of PsMYB62 is 942 bp,which encodes 313 amino acids(aa)and belongs to the R2R3 MYB transcription factor.The plant overexpression vector PBI121-PsMYB62-GFP was constructed and successfully transferred into A.thaliana.The relative expression level of PsMYB62 was significantly increased by CdCl_(2),NaCl,ABA,and mannitol treatments.The germination rate of transgenic seeds was higher than those of wild type(WT)and empty vector(EV)under different concentrations of cadmium treatment.Upon treatment with 100μmol·L^(−1)of CdCl_(2)·2.5H_(2)O,the activities of superoxide dismutase(SOD),peroxidase(POD),and catalase(CAT)in the transgenic plants were significantly higher than those in the WT and EV.The contents of H_(2)O_(2),O_(2)·−and malondialdehyde(MDA)in transgenic lines were increased,but lower than those in WT and EV.The expression levels of AtGSH,AtPCS,and AtNAS4 that were related to the regulation of cadmium were increased,but the expression levels of transgenic lines were higher than those of WT and EV.Conclusion:The above results showed that PsMYB62 could be induced by cadmium and could improve the cadmium resistance of plants.展开更多
For the purpose of functional validation,the mung bean(Vigna radiata)VrPR gene was cloned and overexpressed in Arabidopsis thaliana.Thefindings revealed that the ORF of VrPR contained 1200 bp,in which 399 amino acids w...For the purpose of functional validation,the mung bean(Vigna radiata)VrPR gene was cloned and overexpressed in Arabidopsis thaliana.Thefindings revealed that the ORF of VrPR contained 1200 bp,in which 399 amino acids were encoded.Bioinformatics analysis showed that the VrPR protein belonged to the NADB Rossmann superfamily,which was one of the non-transmembrane hydrophilic proteins.VrPR was assumed to have 44 amino acid phosphorylation sites and be contained in chloroplasts.The VrPR secondary structure comprised of random coil,αhelix,βangle,and extended chain,all of which were quite compatible with the anticipated tertiary structure.Moreover,analysis of the phylogenetic tree indicated that the soybean PR(Glyma.12G222200)and VrPR were closely related.Furthermore,chlorophyll content in leaves is markedly increased in Arabidopsis when VrPR is overexpressed.Ourfindings will serve as a reference for more functional studies on the PR genes in mung bean.展开更多
[Objectives]To clone and analyze the vscB gene of Vibrio alginolyticus HY9901 by bioinformatics.[Methods]A pair of specific primers were designed according to the vscB gene sequence of Vibrio alginolyticus HY9901.The ...[Objectives]To clone and analyze the vscB gene of Vibrio alginolyticus HY9901 by bioinformatics.[Methods]A pair of specific primers were designed according to the vscB gene sequence of Vibrio alginolyticus HY9901.The full length of the primers was cloned by PCR and analyzed by bioinformatics.[Results]The vscB gene was 429 bp long,encoding 142 amino acids,with a theoretical molecular weight of 16.4 kDa and a pI value of 5.48.Amino acid sequence analysis of VscB showed that VscB was not a secretory protein,without signal peptide and transmembrane region,and there were protein kinase C phosphorylation site and casein kinase II phosphorylation site in the sequence.Homologous comparison of amino acid sequences showed that VscB of V.alginolyticus had the highest protein similarity with Vibrio Parahaemolyticus,reaching 91%.Phylogenetic tree analysis showed that the corresponding proteins of V.alginolyticus VscB,Vibrio Parahaemolyticus and Vibrio diabolicus were clustered in the same subfamily.Functional domain analysis showed that it had CesT family domain.Tertiary structure prediction showed that there were 3α-helices and 5β-turns in VscB protein.[Conclusions]This study provided a theoretical basis for further study on the function of chaperone of V.alginolyticus.展开更多
INTRODUCTIONThe liver is one of the organs,which have potentialregenerative capability in mammalian animal.The study of the canine model indicated that theliver could regenerate to original size after 70%hepatectomy i...INTRODUCTIONThe liver is one of the organs,which have potentialregenerative capability in mammalian animal.The study of the canine model indicated that theliver could regenerate to original size after 70%hepatectomy in only two weeks.So it is a hotresearch topic for the cellular and molecularmechanism of liver regeneration.展开更多
AIM: To clone the cDNA of UGT1 A9 from a Chinese human liver and establish the Chinese hamster lung (CHL) cell line expressing human UGTI A9.METHODS: cDNA of UGT1A9 was transcripted from mRNA by reverse transcriptase-...AIM: To clone the cDNA of UGT1 A9 from a Chinese human liver and establish the Chinese hamster lung (CHL) cell line expressing human UGTI A9.METHODS: cDNA of UGT1A9 was transcripted from mRNA by reverse transcriptase-ploymerase chain reaction, and was cloned into the pGEM-T vector which was amplified in the host bacteric E. CoIl DH5α. The inserted fragment, verified by DNA sequencing, wes subcloned into the Hind III/Not I site of a mammalian expression vector pREP9 to construct the plasmid termed pREP9-UGT1A9. CHL cells were transfected with the resultant recombinants, pREP9- UGT1A9, and selected by G418 (400 mg. L-1) for one month. The surviving clone (CHL-UGT1A9) was harvested as a pool and sub-cultured in medium containing G418 to obtain samples for UGT1 A9 assays. The enzyme activity of CHL-UGT1A9 towards propranolol in S9 protein of the cell was determined by HPLC.RESULTS: The sequence of the cDNA segment cloned,which was 1666 bp in length, was identical to that released by Gene Bank (GenBank accession number: AF056188) in coding region. The recombinant constructed, pREP9- UGT1 A9, contains the entire coding region, along with 18bp of the 5' and 55 bp of the 3′ untranslated region of the UGT1A9 cDNA, respectively. The cell lines established expressed the protein of UGT1 A9, and the enzyme activity towards propranolol in S9 protein was found to be 101 ± 24pmol.min-1 .mg-1 protein (n = 3), but was not detectable in parental CHL cells.CONCLUSION: The cDNA of UGT1A9 was successfully cloned from a Chinese human liver and transfected into CHL ceils. The CHL-UGT1A9 cell lines established efficiently expressed the protein of UGT1A9 for the further enzyme study of drug glucuronidation.展开更多
INTRODUCTIONThe mechanism of hepatocellular carcinoma(HCC)is still unclear,although some genes have been found to play a role in the transformation of liver cells,and a variety of studies have described differences in...INTRODUCTIONThe mechanism of hepatocellular carcinoma(HCC)is still unclear,although some genes have been found to play a role in the transformation of liver cells,and a variety of studies have described differences in gene expression which distinguished tumor from nontumor[1-6].The new genes,especially the functional genes directly related with tumor are still worth being found.The purpose of our study is to find the different genes between human liver tumor and normal tissues using suppression subtractive hybridization.展开更多
κ-carrageenan oligosaccharides exhibit various biological activities. Enzymatic degradation by κ-carrageenase is safe and controllable. Therefore, κ-carrageenases have captured more and more attentions. In this stu...κ-carrageenan oligosaccharides exhibit various biological activities. Enzymatic degradation by κ-carrageenase is safe and controllable. Therefore, κ-carrageenases have captured more and more attentions. In this study, a κ-carrageenase encoding gene, cgk X, was cloned from Pseudoalteromonas sp. QY203 with degenerate and inverse PCR. It comprised an ORF of 1194 bp in length, encoding a protein with 397 amino acid residues. Cgk X is a new member of glycoside hydrolase family 16. The deduced amino acid sequence shared a high similarity with Cgk X of Pseudoalteromonas κ-carrageenase; however, the recombinant Cgk X showed different biochemical characteristics. The recombinant enzyme was most active at p H 7.0 and 55℃ in the presence of 300 mmol L^(^(-1))Na Cl. It was stable in a broad range of acidity ranging from p H 3.0 to p H 10.0 when temperature was below 40℃. More than 80% of its activity was maintained after being incubated at p H 3.6–10.0 and 4℃ for 24 h. Cgk X retained more than 90% of activity after being incubated at 40℃ for 1 h. EDTA and SDS(1 mmol L^(-1)) did not inhibit its activity. Cgk X hydrolyzed κ-carrageenan into disaccharide and tetrasaccharide as an endo-cleaver. All these characteristics demonstrated that Cgk X is applicable to both κ-carrageenan oligosaccharide production and κ-carrageenase structure-function research.展开更多
Dendrobium officinale is one of the most precious medicinal plants in China. Its main medicinal ingredients are its secondary metabolites. However,it has the characteristics of limited sources,low active ingredient co...Dendrobium officinale is one of the most precious medicinal plants in China. Its main medicinal ingredients are its secondary metabolites. However,it has the characteristics of limited sources,low active ingredient content and high cost,limiting the use of D. officinale.Studying the network structure and rate-limiting steps of secondary metabolites of medicinal components of D. officinale,analyzing the secondary metabolic synthesis process,mastering the production rules of its medicinal components and carrying out gene cloning or biosynthesis,etc.are of great significance for the rational development and utilization of D. officinale resources. This paper briefly reviews the progress of the research on the secondary metabolites of D. officinale,including the detection and identification of metabolites and the identification and cloning of key metabolic enzymes.展开更多
Summary: Rat transforming growth factor β1 (rTGFβ1) cDNA from rat lymphocytes was cloned by RT-PCR and inserted into pcDNA3 to construct an eukaryotic expression vector, which was named pcDNA3-TGFβ1. The cloned gen...Summary: Rat transforming growth factor β1 (rTGFβ1) cDNA from rat lymphocytes was cloned by RT-PCR and inserted into pcDNA3 to construct an eukaryotic expression vector, which was named pcDNA3-TGFβ1. The cloned gene was confirmed to code rat TGFβ1 by restriction enzyme analysis. pcDNA3-TGFβ1 plasmid was transfected into rat osteoblasts by using liposome-mediated gene transfer technique and the expression of TGFβ1 was detected by using irnmunohistochemical staining assay. It was found that the rat TGFβ1 expression product was obviously detectable in the transfected osteoblasts in 48 h. High expression of TGFβ1 was obtained in the rat osteoblasts in which the constructed TGFβ1 expression vector was transfected.展开更多
Fiber initiation and early elongation areimportant developmental stages at which thefinal fiber number per seed is determined and thefibers show dramatically changes in cell shapeand gene expression.In order to identi...Fiber initiation and early elongation areimportant developmental stages at which thefinal fiber number per seed is determined and thefibers show dramatically changes in cell shapeand gene expression.In order to identify genesfunction in fiber initiation and elongation,cDNA-AFLP technique was used to compare thegene expressions of the ovules of展开更多
Powdery mildew, caused by the biotrophic fungus Blumeria graminis f. sp. tritici(Bgt), is a prevalent disease in common wheat(Triticum aestivum L.) and causes serious yield losses worldwide. We used a map-based approa...Powdery mildew, caused by the biotrophic fungus Blumeria graminis f. sp. tritici(Bgt), is a prevalent disease in common wheat(Triticum aestivum L.) and causes serious yield losses worldwide. We used a map-based approach to clone the major broad-spectrum powdery mildew resistance gene Pm CH1357 from wheat breeding line CH1357. Pm CH1357 was mapped to a 526 kb region containing only Traes CS5 D01 G044600. The Traes CS5 D01 G044600 sequence of the susceptibility allele in Taichung 29(TC29) was identical to that in Chinese Spring, whereas the sequence of the resistance allele in CH1357 was identical to Pm2a previously cloned from the germplasm Ulka/*8Cc. The susceptibility allele in TC29 contained a 7 bp deletion in exon 1, resulting in loss of 856 of the 1277 amino acids in the predicted nucleotide-binding domain leucine-rich repeat containing Pm2a protein.Pm CH1357/Pm2a sequence was also isolated from the Chinese wheat landraces and cultivars that were previously reported to possess the resistance gene Pm2b, Pm2c,PmLX66, or PmND399. The Pm CH1357/Pm2a resistance allele was present in 10 of 495 accessions in core germplasm and contemporary cultivars from China and the USA. A newly developed diagnostic marker for the 7 bp In Del in the resistance gene can be used to eliminate the susceptibility allele in wheat breeding programs.展开更多
Cloning and sequencing of the genes coding for the α and β subunit of phycoerythrin (PE) of a red alga- Gracilaria lemaneiformis (GL) are reported. Alignment of 1084 nucleotides sequenced with three known red algal ...Cloning and sequencing of the genes coding for the α and β subunit of phycoerythrin (PE) of a red alga- Gracilaria lemaneiformis (GL) are reported. Alignment of 1084 nucleotides sequenced with three known red algal PE genes, Rhodella violacea (RV), Polysiphonia boldii (PB) and Aglaothamnion neglectum (AN), showed high level of conservation, and similarities of 77.6% (between GL and RV), 77.9% (GL and AN) and 79.0% (GL and PB). The similarities of amino acids were 84.8% (between GL and RV), 85.7% (GL and PB), and 80.6% (AN and GL), higher than those among nucleotides.展开更多
The genes coding for the α-and β-subunit of allophycocyanin (apcA and apcB) fromthe cyanophyte Spirulina maxima were cloned and sequenced. The results revealed 44.4% of nucleotidesequence similarity and 30.4% of sim...The genes coding for the α-and β-subunit of allophycocyanin (apcA and apcB) fromthe cyanophyte Spirulina maxima were cloned and sequenced. The results revealed 44.4% of nucleotidesequence similarity and 30.4% of similarity of deduced amino acid sequence between them. The aminoacid sequence identities between S. maxima and S. platensis are 99 .4% for α subunit and 100% for βsubunit.展开更多
According to the sequence of the bile salt hydrolase (BSH) gene of Bifidobacterium and the restriction enzyme cutting sites of expression vector pNZ8148, primers were designed and the bile salt hydrolase (BSH) gene wa...According to the sequence of the bile salt hydrolase (BSH) gene of Bifidobacterium and the restriction enzyme cutting sites of expression vector pNZ8148, primers were designed and the bile salt hydrolase (BSH) gene was gotten from Bacillus bifidus ATCC 29521 by PCR. BSH gene was inserted into lactic acid bacteria expression vector pNZ8148 to construct the recombinant pNZ8148-BSH. The recombinant pNZ8148-BSH was transferred into lactic acid bacteria NZ9000 with electrotransformation method. And the recombinant which could express BSH protein was obtained. It was identified by SDS-PAGE electrophoresis and activity verification. The result could provide a rationale reference for expressing BSH in lactic acid bacteria.展开更多
Objective:To clone the virB12 gene in pET28a expression vector for production of recombinant protein to be used as antigenic component for future serological test development.Methods: Brucella melitensis(B.melitensis)...Objective:To clone the virB12 gene in pET28a expression vector for production of recombinant protein to be used as antigenic component for future serological test development.Methods: Brucella melitensis(B.melitensis) 16M strain was cultured and bacterial DNA was extracted by Bioneer AccuPrep~ Genomic DNA Extraction Kit.Oligonucleotide primer pair was designed based on Brucella virB12 gene sequence with BamHI and HindIII restriction site at 5’ end of the forward and reverse primers,respectively.DNA amplification was performed using PrimSTAR~ HS DNA polymerase and the PCK product was purified by DNA AccuPrepGel Purification Kit.Purified DNA was cloned into pJET1.2 cloning vector.VirB12 gene fragment was excised from pJET1.2 asing BamHI/HindIII and subsequendy subcloned into pET28a(+).Results:Brucella virB12 gene was successfully cloned in pJET1.2 and then in pET28a(+) plasmids.PCR and restriction enzyme digestion confirms the procedure.Conclusion:We cloned and expressed the Brucella virB12 gene which could be used as antigenic component for specific serological assay development.展开更多
AIM To obtain human and murine cDNAsencoding IFN--y inducible protein 10 (lP--10) andcytokine responsive gene--2 (Crg-2).METHODS The encoding genes of lP-10 and Crg2 were amplified by RT--PCR from Cultured humanfibrob...AIM To obtain human and murine cDNAsencoding IFN--y inducible protein 10 (lP--10) andcytokine responsive gene--2 (Crg-2).METHODS The encoding genes of lP-10 and Crg2 were amplified by RT--PCR from Cultured humanfibroblast cells and Balb/ c mouse liver treatedby IFN-y and TNF-a, respectively, and clonedinto plasmids of PUC19 and PGEM3Zf(+ ).RESULTS The nucleotide sequences ot theamplified DNA were confirmed by endonucleasesdigestion and sequencing.CONCLUSION Recombinant lP-10/ crg--2 geneclones with 306 hp and 314 hp inserts wereestablished for further research on biologicalactivities and ligands of hiP-10/mCrg--2.展开更多
We cloned and expressed bile salt hydrolase gene of Lactobacillus plantarum M1-UVS29 in Lactococcus lactis NZ9000 successfully. Gene-specific primers for amplification of L. plantarum bsh were designed by using sequen...We cloned and expressed bile salt hydrolase gene of Lactobacillus plantarum M1-UVS29 in Lactococcus lactis NZ9000 successfully. Gene-specific primers for amplification of L. plantarum bsh were designed by using sequence which availabled from Gen Bank. The production of PCR amplicon was confirmed by sequencing and cloned into pMD18-T vector, and then recombined into expression vector p NZ8148 and yielding vector p NZ8148-BSH. p NZ8148-BSH was transferred into Lactococcus lactis NZ9000. Sequencing indicated that the cloned bsh fragment contained 995 nucleotides, and shared 99.3% sequence homology with bsh gene from L. plantarum MBUL10. Cloned bsh fragment was successfully transduced into NICE expression system and confirmed by PCR and restriction digest. Recombinant BSH protein was analyzed by SDS-PAGE. The molecular weight of BSH protein was approximately 37 ku. Activity of the expressed protein was 0.77 μmol · min-1. The successfully expressed proteins by genetic engineering technology made the function of lactic acid bacteria be abundant and laid the foundation for further researches into cholesterol-lowering lactic acid bacterium food and probiotics.展开更多
基金funded by the National Natural Science Foundation of China(21904139)。
文摘Background:Tumor cell heterogeneity mediated drug resistance has been recognized as the stumbling block of cancer treatment.Elucidating the cytotoxicity of anticancer drugs at single-cell level in a high-throughput way is thus of great value for developing precision therapy.However,current techniques suffer from limitations in dynamically characterizing the responses of thousands of single cells or cell clones presented to multiple drug conditions.Methods:We developed a new microfluidics-based“SMART”platform that is Simple to operate,able to generate a Massive single-cell array and Multiplex drug concentrations,capable of keeping cells Alive,Retainable and Trackable in the microchambers.These features are achieved by integrating a Microfluidic chamber Array(4320 units)and a sixConcentration gradient generator(MAC),which enables highly efficient analysis of leukemia drug effects on single cells and cell clones in a high-throughput way.Results:A simple procedure produces 6 on-chip drug gradients to treat more than 3000 single cells or single-cell derived clones and thus allows an efficient and precise analysis of cell heterogeneity.The statistic results reveal that Imatinib(Ima)and Resveratrol(Res)combination treatment on single cells or clones is much more efficient than Ima or Res single drug treatment,indicated by the markedly reduced half maximal inhibitory concentration(IC50).Additionally,single-cell derived clones demonstrate a higher IC_(50) in each drug treatment compared to single cells.Moreover,primary cells isolated from two leukemia patients are also found with apparent heterogeneity upon drug treatment on MAC.Conclusions:This microfluidics-based“SMART”platform allows high-throughput single-cell capture and culture,dynamic drug-gradient treatment and cell response monitoring,which represents a new approach to efficiently investigate anticancer drug effects and should benefit drug discovery for leukemia and other cancers.
基金funded by the Natural Science Foundation of Heilongjiang Province(LH2020C045).
文摘Potentilla sericea is a heavy metal hyperaccumulator landscaping plant.MYB transcription factors play an important role in regulating plant stress response to adversity.However,there are few studies on MYB transcription factors in stress tolerance in Potentilla sericea.In this study,the PsMYB62 gene was successfully cloned from Potentilla sericea.Methods:Bioinformatic analysis and real-time quantitative PCR(qPCR)methods were used to evaluate this gene.The transgenic A.thaliana were obtained by flower dipping and the gene function was identified by determining physiological indicators under cadmium stress.Results:The open reading frame of PsMYB62 is 942 bp,which encodes 313 amino acids(aa)and belongs to the R2R3 MYB transcription factor.The plant overexpression vector PBI121-PsMYB62-GFP was constructed and successfully transferred into A.thaliana.The relative expression level of PsMYB62 was significantly increased by CdCl_(2),NaCl,ABA,and mannitol treatments.The germination rate of transgenic seeds was higher than those of wild type(WT)and empty vector(EV)under different concentrations of cadmium treatment.Upon treatment with 100μmol·L^(−1)of CdCl_(2)·2.5H_(2)O,the activities of superoxide dismutase(SOD),peroxidase(POD),and catalase(CAT)in the transgenic plants were significantly higher than those in the WT and EV.The contents of H_(2)O_(2),O_(2)·−and malondialdehyde(MDA)in transgenic lines were increased,but lower than those in WT and EV.The expression levels of AtGSH,AtPCS,and AtNAS4 that were related to the regulation of cadmium were increased,but the expression levels of transgenic lines were higher than those of WT and EV.Conclusion:The above results showed that PsMYB62 could be induced by cadmium and could improve the cadmium resistance of plants.
基金This research was funded by National Natural Science Foundation of China,Grant Number(31871576)National Keypoint Research and Invention Program of the Thirteenth,Grant Number(2019YFD1002205)The APC was funded by National Keypoint Research and Invention Program of the Thirteenth.
文摘For the purpose of functional validation,the mung bean(Vigna radiata)VrPR gene was cloned and overexpressed in Arabidopsis thaliana.Thefindings revealed that the ORF of VrPR contained 1200 bp,in which 399 amino acids were encoded.Bioinformatics analysis showed that the VrPR protein belonged to the NADB Rossmann superfamily,which was one of the non-transmembrane hydrophilic proteins.VrPR was assumed to have 44 amino acid phosphorylation sites and be contained in chloroplasts.The VrPR secondary structure comprised of random coil,αhelix,βangle,and extended chain,all of which were quite compatible with the anticipated tertiary structure.Moreover,analysis of the phylogenetic tree indicated that the soybean PR(Glyma.12G222200)and VrPR were closely related.Furthermore,chlorophyll content in leaves is markedly increased in Arabidopsis when VrPR is overexpressed.Ourfindings will serve as a reference for more functional studies on the PR genes in mung bean.
基金Supported by Outstanding Graduate Entering Laboratory Project of College of Fisheries,Guangdong Ocean UniversityGrants from Shenzhen Science and Technology Project(JCYJ20190813104207152)+3 种基金National Natural Science Foundation of China(32073015)Natural Science Foundation of Guangdong Province(2021A1515011078)Undergraduate Innovation and Entrepreneurship Training Program of Guangdong Ocean University(CXXL2022005)Undergraduate Innovation Team of Guangdong Ocean University(CCTD201802).
文摘[Objectives]To clone and analyze the vscB gene of Vibrio alginolyticus HY9901 by bioinformatics.[Methods]A pair of specific primers were designed according to the vscB gene sequence of Vibrio alginolyticus HY9901.The full length of the primers was cloned by PCR and analyzed by bioinformatics.[Results]The vscB gene was 429 bp long,encoding 142 amino acids,with a theoretical molecular weight of 16.4 kDa and a pI value of 5.48.Amino acid sequence analysis of VscB showed that VscB was not a secretory protein,without signal peptide and transmembrane region,and there were protein kinase C phosphorylation site and casein kinase II phosphorylation site in the sequence.Homologous comparison of amino acid sequences showed that VscB of V.alginolyticus had the highest protein similarity with Vibrio Parahaemolyticus,reaching 91%.Phylogenetic tree analysis showed that the corresponding proteins of V.alginolyticus VscB,Vibrio Parahaemolyticus and Vibrio diabolicus were clustered in the same subfamily.Functional domain analysis showed that it had CesT family domain.Tertiary structure prediction showed that there were 3α-helices and 5β-turns in VscB protein.[Conclusions]This study provided a theoretical basis for further study on the function of chaperone of V.alginolyticus.
文摘INTRODUCTIONThe liver is one of the organs,which have potentialregenerative capability in mammalian animal.The study of the canine model indicated that theliver could regenerate to original size after 70%hepatectomy in only two weeks.So it is a hotresearch topic for the cellular and molecularmechanism of liver regeneration.
基金Supported by the National Natural Science Foundation of China(C39370805)Zhejiang Provincial Natural Science Foundation(300487)the Excellent Youth Scientist Fund of Zhejiang Province
文摘AIM: To clone the cDNA of UGT1 A9 from a Chinese human liver and establish the Chinese hamster lung (CHL) cell line expressing human UGTI A9.METHODS: cDNA of UGT1A9 was transcripted from mRNA by reverse transcriptase-ploymerase chain reaction, and was cloned into the pGEM-T vector which was amplified in the host bacteric E. CoIl DH5α. The inserted fragment, verified by DNA sequencing, wes subcloned into the Hind III/Not I site of a mammalian expression vector pREP9 to construct the plasmid termed pREP9-UGT1A9. CHL cells were transfected with the resultant recombinants, pREP9- UGT1A9, and selected by G418 (400 mg. L-1) for one month. The surviving clone (CHL-UGT1A9) was harvested as a pool and sub-cultured in medium containing G418 to obtain samples for UGT1 A9 assays. The enzyme activity of CHL-UGT1A9 towards propranolol in S9 protein of the cell was determined by HPLC.RESULTS: The sequence of the cDNA segment cloned,which was 1666 bp in length, was identical to that released by Gene Bank (GenBank accession number: AF056188) in coding region. The recombinant constructed, pREP9- UGT1 A9, contains the entire coding region, along with 18bp of the 5' and 55 bp of the 3′ untranslated region of the UGT1A9 cDNA, respectively. The cell lines established expressed the protein of UGT1 A9, and the enzyme activity towards propranolol in S9 protein was found to be 101 ± 24pmol.min-1 .mg-1 protein (n = 3), but was not detectable in parental CHL cells.CONCLUSION: The cDNA of UGT1A9 was successfully cloned from a Chinese human liver and transfected into CHL ceils. The CHL-UGT1A9 cell lines established efficiently expressed the protein of UGT1A9 for the further enzyme study of drug glucuronidation.
文摘INTRODUCTIONThe mechanism of hepatocellular carcinoma(HCC)is still unclear,although some genes have been found to play a role in the transformation of liver cells,and a variety of studies have described differences in gene expression which distinguished tumor from nontumor[1-6].The new genes,especially the functional genes directly related with tumor are still worth being found.The purpose of our study is to find the different genes between human liver tumor and normal tissues using suppression subtractive hybridization.
基金supported by the National High-Tech R&D Program(No.2011AA090703)the National Natural Science Foundation of China(No.31070712)the Special Fund for Marine Scientific Research in the Public Interest(Nos.201105027 and 201005024)
文摘κ-carrageenan oligosaccharides exhibit various biological activities. Enzymatic degradation by κ-carrageenase is safe and controllable. Therefore, κ-carrageenases have captured more and more attentions. In this study, a κ-carrageenase encoding gene, cgk X, was cloned from Pseudoalteromonas sp. QY203 with degenerate and inverse PCR. It comprised an ORF of 1194 bp in length, encoding a protein with 397 amino acid residues. Cgk X is a new member of glycoside hydrolase family 16. The deduced amino acid sequence shared a high similarity with Cgk X of Pseudoalteromonas κ-carrageenase; however, the recombinant Cgk X showed different biochemical characteristics. The recombinant enzyme was most active at p H 7.0 and 55℃ in the presence of 300 mmol L^(^(-1))Na Cl. It was stable in a broad range of acidity ranging from p H 3.0 to p H 10.0 when temperature was below 40℃. More than 80% of its activity was maintained after being incubated at p H 3.6–10.0 and 4℃ for 24 h. Cgk X retained more than 90% of activity after being incubated at 40℃ for 1 h. EDTA and SDS(1 mmol L^(-1)) did not inhibit its activity. Cgk X hydrolyzed κ-carrageenan into disaccharide and tetrasaccharide as an endo-cleaver. All these characteristics demonstrated that Cgk X is applicable to both κ-carrageenan oligosaccharide production and κ-carrageenase structure-function research.
基金Supported by National Natural Science Foundation of China(81060028)Key Project of Science and Technology of Guangxi Zhuang Autonomous Region(Gui Ke Zhong 1598005-9)Key Science and Technology Program of Guangxi Zhuang Autonomous Region(Gui Ke Gong 1355005-3-7)
文摘Dendrobium officinale is one of the most precious medicinal plants in China. Its main medicinal ingredients are its secondary metabolites. However,it has the characteristics of limited sources,low active ingredient content and high cost,limiting the use of D. officinale.Studying the network structure and rate-limiting steps of secondary metabolites of medicinal components of D. officinale,analyzing the secondary metabolic synthesis process,mastering the production rules of its medicinal components and carrying out gene cloning or biosynthesis,etc.are of great significance for the rational development and utilization of D. officinale resources. This paper briefly reviews the progress of the research on the secondary metabolites of D. officinale,including the detection and identification of metabolites and the identification and cloning of key metabolic enzymes.
文摘Summary: Rat transforming growth factor β1 (rTGFβ1) cDNA from rat lymphocytes was cloned by RT-PCR and inserted into pcDNA3 to construct an eukaryotic expression vector, which was named pcDNA3-TGFβ1. The cloned gene was confirmed to code rat TGFβ1 by restriction enzyme analysis. pcDNA3-TGFβ1 plasmid was transfected into rat osteoblasts by using liposome-mediated gene transfer technique and the expression of TGFβ1 was detected by using irnmunohistochemical staining assay. It was found that the rat TGFβ1 expression product was obviously detectable in the transfected osteoblasts in 48 h. High expression of TGFβ1 was obtained in the rat osteoblasts in which the constructed TGFβ1 expression vector was transfected.
文摘Fiber initiation and early elongation areimportant developmental stages at which thefinal fiber number per seed is determined and thefibers show dramatically changes in cell shapeand gene expression.In order to identify genesfunction in fiber initiation and elongation,cDNA-AFLP technique was used to compare thegene expressions of the ovules of
基金supported by funding from the National Key Research and Development Program of China (2016YFD0102000)Shanxi Provincial Key Platform for Science and Technology Innovation Program (201605D151002)+2 种基金Shanxi Provincial International Cooperation in Science and Technology Program (201803D221018-5)Shanxi Provincial Key Research and Development Project (201803D421020 and 201703D211007)Shanxi Academy of Agricultural Sciences (Project YGG17123)
文摘Powdery mildew, caused by the biotrophic fungus Blumeria graminis f. sp. tritici(Bgt), is a prevalent disease in common wheat(Triticum aestivum L.) and causes serious yield losses worldwide. We used a map-based approach to clone the major broad-spectrum powdery mildew resistance gene Pm CH1357 from wheat breeding line CH1357. Pm CH1357 was mapped to a 526 kb region containing only Traes CS5 D01 G044600. The Traes CS5 D01 G044600 sequence of the susceptibility allele in Taichung 29(TC29) was identical to that in Chinese Spring, whereas the sequence of the resistance allele in CH1357 was identical to Pm2a previously cloned from the germplasm Ulka/*8Cc. The susceptibility allele in TC29 contained a 7 bp deletion in exon 1, resulting in loss of 856 of the 1277 amino acids in the predicted nucleotide-binding domain leucine-rich repeat containing Pm2a protein.Pm CH1357/Pm2a sequence was also isolated from the Chinese wheat landraces and cultivars that were previously reported to possess the resistance gene Pm2b, Pm2c,PmLX66, or PmND399. The Pm CH1357/Pm2a resistance allele was present in 10 of 495 accessions in core germplasm and contemporary cultivars from China and the USA. A newly developed diagnostic marker for the 7 bp In Del in the resistance gene can be used to eliminate the susceptibility allele in wheat breeding programs.
文摘Cloning and sequencing of the genes coding for the α and β subunit of phycoerythrin (PE) of a red alga- Gracilaria lemaneiformis (GL) are reported. Alignment of 1084 nucleotides sequenced with three known red algal PE genes, Rhodella violacea (RV), Polysiphonia boldii (PB) and Aglaothamnion neglectum (AN), showed high level of conservation, and similarities of 77.6% (between GL and RV), 77.9% (GL and AN) and 79.0% (GL and PB). The similarities of amino acids were 84.8% (between GL and RV), 85.7% (GL and PB), and 80.6% (AN and GL), higher than those among nucleotides.
文摘The genes coding for the α-and β-subunit of allophycocyanin (apcA and apcB) fromthe cyanophyte Spirulina maxima were cloned and sequenced. The results revealed 44.4% of nucleotidesequence similarity and 30.4% of similarity of deduced amino acid sequence between them. The aminoacid sequence identities between S. maxima and S. platensis are 99 .4% for α subunit and 100% for βsubunit.
基金Supported by 863 Projects (2008AA10Z311)National Science and Technology Support Projects (2009BADB9B06)+1 种基金Started Post-doctoral Research Grant of Heilongjiang Province (LBH-Q07023)Harbin Technological Innovation of Special Funds (2007RFQXN020)
文摘According to the sequence of the bile salt hydrolase (BSH) gene of Bifidobacterium and the restriction enzyme cutting sites of expression vector pNZ8148, primers were designed and the bile salt hydrolase (BSH) gene was gotten from Bacillus bifidus ATCC 29521 by PCR. BSH gene was inserted into lactic acid bacteria expression vector pNZ8148 to construct the recombinant pNZ8148-BSH. The recombinant pNZ8148-BSH was transferred into lactic acid bacteria NZ9000 with electrotransformation method. And the recombinant which could express BSH protein was obtained. It was identified by SDS-PAGE electrophoresis and activity verification. The result could provide a rationale reference for expressing BSH in lactic acid bacteria.
基金supported by grant from Tehran University of Medical Sciences
文摘Objective:To clone the virB12 gene in pET28a expression vector for production of recombinant protein to be used as antigenic component for future serological test development.Methods: Brucella melitensis(B.melitensis) 16M strain was cultured and bacterial DNA was extracted by Bioneer AccuPrep~ Genomic DNA Extraction Kit.Oligonucleotide primer pair was designed based on Brucella virB12 gene sequence with BamHI and HindIII restriction site at 5’ end of the forward and reverse primers,respectively.DNA amplification was performed using PrimSTAR~ HS DNA polymerase and the PCK product was purified by DNA AccuPrepGel Purification Kit.Purified DNA was cloned into pJET1.2 cloning vector.VirB12 gene fragment was excised from pJET1.2 asing BamHI/HindIII and subsequendy subcloned into pET28a(+).Results:Brucella virB12 gene was successfully cloned in pJET1.2 and then in pET28a(+) plasmids.PCR and restriction enzyme digestion confirms the procedure.Conclusion:We cloned and expressed the Brucella virB12 gene which could be used as antigenic component for specific serological assay development.
基金Supported by the 0utstanding Youth Fund from National Natural Science Foundation of China,No.39625023
文摘AIM To obtain human and murine cDNAsencoding IFN--y inducible protein 10 (lP--10) andcytokine responsive gene--2 (Crg-2).METHODS The encoding genes of lP-10 and Crg2 were amplified by RT--PCR from Cultured humanfibroblast cells and Balb/ c mouse liver treatedby IFN-y and TNF-a, respectively, and clonedinto plasmids of PUC19 and PGEM3Zf(+ ).RESULTS The nucleotide sequences ot theamplified DNA were confirmed by endonucleasesdigestion and sequencing.CONCLUSION Recombinant lP-10/ crg--2 geneclones with 306 hp and 314 hp inserts wereestablished for further research on biologicalactivities and ligands of hiP-10/mCrg--2.
基金Supported by the National Natural Science Fund Project(31171657)Heilongjiang Province Natural Fund Project(ZD201207)Heilongjiang Province Postdoctoral Special Funds(LBH-Q13133)
文摘We cloned and expressed bile salt hydrolase gene of Lactobacillus plantarum M1-UVS29 in Lactococcus lactis NZ9000 successfully. Gene-specific primers for amplification of L. plantarum bsh were designed by using sequence which availabled from Gen Bank. The production of PCR amplicon was confirmed by sequencing and cloned into pMD18-T vector, and then recombined into expression vector p NZ8148 and yielding vector p NZ8148-BSH. p NZ8148-BSH was transferred into Lactococcus lactis NZ9000. Sequencing indicated that the cloned bsh fragment contained 995 nucleotides, and shared 99.3% sequence homology with bsh gene from L. plantarum MBUL10. Cloned bsh fragment was successfully transduced into NICE expression system and confirmed by PCR and restriction digest. Recombinant BSH protein was analyzed by SDS-PAGE. The molecular weight of BSH protein was approximately 37 ku. Activity of the expressed protein was 0.77 μmol · min-1. The successfully expressed proteins by genetic engineering technology made the function of lactic acid bacteria be abundant and laid the foundation for further researches into cholesterol-lowering lactic acid bacterium food and probiotics.
