Curcin, a ribosome-inactivating protein with a molecular weight of about 28.2 kD, which strongly inhibits the protein synthesis in rabbit reticulocyte lysate system with an IC50 value of about (0.19 +/- 0.01) nmol/L, ...Curcin, a ribosome-inactivating protein with a molecular weight of about 28.2 kD, which strongly inhibits the protein synthesis in rabbit reticulocyte lysate system with an IC50 value of about (0.19 +/- 0.01) nmol/L, was purified from the seeds of Jatropha curcas L. The protein has the activity of rRNA N-glycosidase. Degenerate primers were designed based on the N-terminal partial sequence from purified curcin. The full-length curcin cDNA by RT-PCR and 5'-RACE was cloned. The deduced amino acids sequence indicates that a preprotein with 20 amino acid residues is first translated and then processed to a mature protein with 251 amino acids. The deduced amino acids sequence shares homology of 33% and 57% to those of type I ribosome-inactivating proteins (RIPs) and A chain of type II RIPs, respectively. The sequence encoding mature curcin was integrated into the pQE-30 vector for expression in Escherichia coli strain M15 (pREP4). The purified recombinant curcin was able to inhibit protein synthesis in rabbit reticulocyte lysate system.展开更多
[Objective] The aim is to study the construction of vectors expressing foreign protein in potato starch grains specifically, and provide some reference for solving industrialized core problem of high cost and low expr...[Objective] The aim is to study the construction of vectors expressing foreign protein in potato starch grains specifically, and provide some reference for solving industrialized core problem of high cost and low expression level of foreign protein. [Method] By using molecular biological techniques of RT-PCR and nested PCR, plant expression vector for the foreign protein locating in the potato starch grains was constructed. [ Result] Coding sequence （ GC20 ） of potato starch grains that was located and expressed by GBSSI promoter was cloned. Plant expression vector was screened out through connection, transformation and enzyme digestion identification. [ Conclusion] This result laid a foundation for further screening the foreign protein on the potato starch grains.展开更多
[Objective] To construct prokaryotic expression vectors encoding gene Erb3binding protein （EBP1）, which plays important roles in regulating plant organ size from Nervilia fordii （Hance） Schltr. [Methods] PCR produ...[Objective] To construct prokaryotic expression vectors encoding gene Erb3binding protein （EBP1）, which plays important roles in regulating plant organ size from Nervilia fordii （Hance） Schltr. [Methods] PCR products of NfEBP1 with particular restriction sites and expression vectors, pET-28 and pET-16b were digested. Ligation, transformation and selection were performed to construct the recombinant plasmids pET-28-NfEBP1 and pET-16-NfEBP1. The recombinant plasmids were transformed into E. coli BL21 using heat -shock transformation. [Results] Recombinant plasmids pET-28-NfEBP1-1188 and pET-16-NfEBP1-1188 were constructed and transformed into expressional host cells, E. coli BL21, and validated by colony PCR, sequencing and double digestion. [Conclusion] Prokaryotic expression vectors of EBP1 gene from N. fordii were successfully constructed, which laid the foundation for characterization of the gene function.展开更多
Pea ( Pisum sativum Linn.) actin gene family contains at least three isoforms (PEAcⅠ, PEAcⅡand PEAcⅢ), and the DNA sequence of these isoforms show high similarity in the coding regions and significant divergence...Pea ( Pisum sativum Linn.) actin gene family contains at least three isoforms (PEAcⅠ, PEAcⅡand PEAcⅢ), and the DNA sequence of these isoforms show high similarity in the coding regions and significant divergence in the untranslated regions. RT_PCR and Southern blotting using 3′_untranslated region (3′_UTR) as specific probe revealed that pea isoactin genes were expressed in roots, stems, leaves, tendrils, pollen and juvenile siliques, but displayed different patterns of transcript accumulation. Two_fold serial dilution electrophoresis showed PEAcⅠ mRNA preferentially accumulated in rapidly developing tissues: it peaked in seven days' stem; remained at a rather high level in leaves within a month but decreased significantly later; varied a little in tendrils and reached a median and a very low level respectively in juvenile siliques and in pollen. PEAcⅡ displayed somewhat similar expression pattern to PEAcⅠ. The observed differences in sequences and transcript accumulation patterns suggest that the individual pea actin genes may differ in their transcriptional regulation and cellular function. Phylogenetic tree of actins showed that pea actin isoforms are as diverged from each other as they are from other plant actins, and pea actins might have originated from a common ancestor before the divergence of the dicot and monocot plants.展开更多
[ Objective] The aim of this study was to investigate the construction and identification of siRNA expression vector targeting nucleocapsid protein N gone of PRRSV. [Method] Three siRNA oligonucleotides targeting nucl...[ Objective] The aim of this study was to investigate the construction and identification of siRNA expression vector targeting nucleocapsid protein N gone of PRRSV. [Method] Three siRNA oligonucleotides targeting nucleocapsid protein N gone sequence of PRRSV were designed or synthesized, and then inserted into CMV promoter downstream to clone into pSilencer 4,1 -CMV eukaryotic expression vector. The recombinant expression vector was identified by enzyme digestion and DNA sequencing. [ Result] The results showed that the siRNA interference recombinant plasmid vector pSilencer-N targeting nucleocapsid protein gone expression had been successfully constructed. [ Conclusion] This study lays a foundation for studies on the controlling PRRSV by RNA interference technique .展开更多
Objective: To construct a recombinant plasmid containing the outer membrane protein 2 (Omp2) gene of Chlamydia trachomatis and express Omp2 in E.coli. Methods: The omp2 gene of C. trachomatis serovar D was cloned into...Objective: To construct a recombinant plasmid containing the outer membrane protein 2 (Omp2) gene of Chlamydia trachomatis and express Omp2 in E.coli. Methods: The omp2 gene of C. trachomatis serovar D was cloned into pQE30 vector following PCR amplification from genomic DNA. E. coli M15 transformants were induced to express the fusion protein by IPTG and the product was identified by SDS-PAGE and Western blot. Results: Confirmed by enzyme cleavage analysis and DNA sequencing, a correct recombinant plasmid pQE30/omp2 was constructed. The fusion protein from the transformants was approximately 60 kDa in size in SDS-PAGE analysis, which could specially react with anti-6 X His mouse monoclonal IgG antibodies. Conclusion: We successfully expressed Omp2 in E. coli M15, providing an efficient and simple system for assaying the immunological properties of Omp2.展开更多
A symptom of chilling injury is development of water deficit in shoots, resulting from an imbalance of water transport and transpiration. In this work, two rice varieties （Oryza sativa L. var. Wasetoitsu and Somewake...A symptom of chilling injury is development of water deficit in shoots, resulting from an imbalance of water transport and transpiration. In this work, two rice varieties （Oryza sativa L. var. Wasetoitsu and Somewake） seedlings were chilled at 7 ℃, followed by recovery at 28 ℃. Based on the growth phenotype and electrolyte leakage tests, Somewake was shown to be a chilling-tolerant variety, and Wasetoitsu a chilling-sensitive one. The chilling stress reduced markedly the relative water content （RWC） of leaves, accumulative transpiration and osmotic root hydraulic conductivity （Lp） in both varieties. But when retumed to 28 ℃, the water relation balance of Somewake recovered better. The mRNA expression profile of all the 11 plasma membrane intrinsic proteins （PIPs）, a subgroup of aquaporins, was subsequently determined by real-time reverse transcription （RT）-PCR with TaqMan-minor grove binder （MGB） probes derived from rice var. Nipponbare during chilling treatment and recovery. Most of the PIP genes was down-regulated at the low temperature, and recovered at the warm temperature. The relative expression of some PIPs in both Somewake and Wasetoitsu decreased in parallel during the chilling. However during the recovery, the relative expression of OsPIP1;1, OsPIP2;1, OsPIP2;7 in shoots and OsPIP1：1, OsPIP2：1 in roots were significantly higher in Somewake than Wasetoitsu. This supports the role of PIPs in re-establishing water balance after chilling conditions. We discuss the diversified roles played by members of the aquaporin PIP subfamily in plant chilling tolerance depending on aquaporin isoforms, plant tissue and the stage of chilling duration.展开更多
文摘Curcin, a ribosome-inactivating protein with a molecular weight of about 28.2 kD, which strongly inhibits the protein synthesis in rabbit reticulocyte lysate system with an IC50 value of about (0.19 +/- 0.01) nmol/L, was purified from the seeds of Jatropha curcas L. The protein has the activity of rRNA N-glycosidase. Degenerate primers were designed based on the N-terminal partial sequence from purified curcin. The full-length curcin cDNA by RT-PCR and 5'-RACE was cloned. The deduced amino acids sequence indicates that a preprotein with 20 amino acid residues is first translated and then processed to a mature protein with 251 amino acids. The deduced amino acids sequence shares homology of 33% and 57% to those of type I ribosome-inactivating proteins (RIPs) and A chain of type II RIPs, respectively. The sequence encoding mature curcin was integrated into the pQE-30 vector for expression in Escherichia coli strain M15 (pREP4). The purified recombinant curcin was able to inhibit protein synthesis in rabbit reticulocyte lysate system.
基金Supported by the National Natural Science Foundation Program(30160010)~~
文摘[Objective] The aim is to study the construction of vectors expressing foreign protein in potato starch grains specifically, and provide some reference for solving industrialized core problem of high cost and low expression level of foreign protein. [Method] By using molecular biological techniques of RT-PCR and nested PCR, plant expression vector for the foreign protein locating in the potato starch grains was constructed. [ Result] Coding sequence （ GC20 ） of potato starch grains that was located and expressed by GBSSI promoter was cloned. Plant expression vector was screened out through connection, transformation and enzyme digestion identification. [ Conclusion] This result laid a foundation for further screening the foreign protein on the potato starch grains.
基金Supported by Research Fund of the Doctoral Program of Higher Education (200805720004)Scientific Research Foundation for Returned Scholars, Ministry of Education of China ([2009]1001)~~
文摘[Objective] To construct prokaryotic expression vectors encoding gene Erb3binding protein （EBP1）, which plays important roles in regulating plant organ size from Nervilia fordii （Hance） Schltr. [Methods] PCR products of NfEBP1 with particular restriction sites and expression vectors, pET-28 and pET-16b were digested. Ligation, transformation and selection were performed to construct the recombinant plasmids pET-28-NfEBP1 and pET-16-NfEBP1. The recombinant plasmids were transformed into E. coli BL21 using heat -shock transformation. [Results] Recombinant plasmids pET-28-NfEBP1-1188 and pET-16-NfEBP1-1188 were constructed and transformed into expressional host cells, E. coli BL21, and validated by colony PCR, sequencing and double digestion. [Conclusion] Prokaryotic expression vectors of EBP1 gene from N. fordii were successfully constructed, which laid the foundation for characterization of the gene function.
文摘Pea ( Pisum sativum Linn.) actin gene family contains at least three isoforms (PEAcⅠ, PEAcⅡand PEAcⅢ), and the DNA sequence of these isoforms show high similarity in the coding regions and significant divergence in the untranslated regions. RT_PCR and Southern blotting using 3′_untranslated region (3′_UTR) as specific probe revealed that pea isoactin genes were expressed in roots, stems, leaves, tendrils, pollen and juvenile siliques, but displayed different patterns of transcript accumulation. Two_fold serial dilution electrophoresis showed PEAcⅠ mRNA preferentially accumulated in rapidly developing tissues: it peaked in seven days' stem; remained at a rather high level in leaves within a month but decreased significantly later; varied a little in tendrils and reached a median and a very low level respectively in juvenile siliques and in pollen. PEAcⅡ displayed somewhat similar expression pattern to PEAcⅠ. The observed differences in sequences and transcript accumulation patterns suggest that the individual pea actin genes may differ in their transcriptional regulation and cellular function. Phylogenetic tree of actins showed that pea actin isoforms are as diverged from each other as they are from other plant actins, and pea actins might have originated from a common ancestor before the divergence of the dicot and monocot plants.
基金Supported by Based on Cuttingedge technology and research Project of Henan Province(072300430060)The focus of Scientific andTechnological Project of Henan Province(072102130023)Colleges and Universities of Henan Province in Support of TechnologicalInnovation Plan~~
文摘[ Objective] The aim of this study was to investigate the construction and identification of siRNA expression vector targeting nucleocapsid protein N gone of PRRSV. [Method] Three siRNA oligonucleotides targeting nucleocapsid protein N gone sequence of PRRSV were designed or synthesized, and then inserted into CMV promoter downstream to clone into pSilencer 4,1 -CMV eukaryotic expression vector. The recombinant expression vector was identified by enzyme digestion and DNA sequencing. [ Result] The results showed that the siRNA interference recombinant plasmid vector pSilencer-N targeting nucleocapsid protein gone expression had been successfully constructed. [ Conclusion] This study lays a foundation for studies on the controlling PRRSV by RNA interference technique .
基金This work was supported in part by grants from the Department of Science and Technology of Hunan Province (No. 01SSY2008-6) the Department of Health of Hunan Province (No. B2003-078).
文摘Objective: To construct a recombinant plasmid containing the outer membrane protein 2 (Omp2) gene of Chlamydia trachomatis and express Omp2 in E.coli. Methods: The omp2 gene of C. trachomatis serovar D was cloned into pQE30 vector following PCR amplification from genomic DNA. E. coli M15 transformants were induced to express the fusion protein by IPTG and the product was identified by SDS-PAGE and Western blot. Results: Confirmed by enzyme cleavage analysis and DNA sequencing, a correct recombinant plasmid pQE30/omp2 was constructed. The fusion protein from the transformants was approximately 60 kDa in size in SDS-PAGE analysis, which could specially react with anti-6 X His mouse monoclonal IgG antibodies. Conclusion: We successfully expressed Omp2 in E. coli M15, providing an efficient and simple system for assaying the immunological properties of Omp2.
文摘A symptom of chilling injury is development of water deficit in shoots, resulting from an imbalance of water transport and transpiration. In this work, two rice varieties （Oryza sativa L. var. Wasetoitsu and Somewake） seedlings were chilled at 7 ℃, followed by recovery at 28 ℃. Based on the growth phenotype and electrolyte leakage tests, Somewake was shown to be a chilling-tolerant variety, and Wasetoitsu a chilling-sensitive one. The chilling stress reduced markedly the relative water content （RWC） of leaves, accumulative transpiration and osmotic root hydraulic conductivity （Lp） in both varieties. But when retumed to 28 ℃, the water relation balance of Somewake recovered better. The mRNA expression profile of all the 11 plasma membrane intrinsic proteins （PIPs）, a subgroup of aquaporins, was subsequently determined by real-time reverse transcription （RT）-PCR with TaqMan-minor grove binder （MGB） probes derived from rice var. Nipponbare during chilling treatment and recovery. Most of the PIP genes was down-regulated at the low temperature, and recovered at the warm temperature. The relative expression of some PIPs in both Somewake and Wasetoitsu decreased in parallel during the chilling. However during the recovery, the relative expression of OsPIP1;1, OsPIP2;1, OsPIP2;7 in shoots and OsPIP1：1, OsPIP2：1 in roots were significantly higher in Somewake than Wasetoitsu. This supports the role of PIPs in re-establishing water balance after chilling conditions. We discuss the diversified roles played by members of the aquaporin PIP subfamily in plant chilling tolerance depending on aquaporin isoforms, plant tissue and the stage of chilling duration.
