The agp gene encoding the ADP-glucose pyrophosphorylase involved in cyanobacterial glycogen synthesis was amplified by PCR. The resulting agp fragment was cloned in plasmid pUC118 to generate plasmid pUCA. Part of the...The agp gene encoding the ADP-glucose pyrophosphorylase involved in cyanobacterial glycogen synthesis was amplified by PCR. The resulting agp fragment was cloned in plasmid pUC118 to generate plasmid pUCA. Part of the fragment within the agp DNA was deleted and replaced by an erythromycin resistance cassette to generate plasmid pUCAE, which was used to transform the Synechocystis sp. PCC 6803 wild-type strain and a mutant with resistance to erythromycin was obtained. PCR analysis of the genomic DNA from the resulting mutant indicated that the appropriate deletion and insertion indeed had occurred. The cell growth and Chl a, glycogen content in the mutant showed difference from those in the wild-type strain. The obtained biomass as well as the Chl a content in the mutant strain was higher than that of the wild-type strain, which suggested that the photosynthesis efficiency in the agp(-) strain was higher than that in the wild-type strain. No glycogen was found in the mutant, providing evidence for the correction of the mutant in physiological level.展开更多
[Objective] The research aimed to reveal the functions of NAC and UBA domains in Peatl＇s thermal stability. [Method] Fusion expression vectors of Pearl protein and the 3 deletion mutants were constructed. The recombi...[Objective] The research aimed to reveal the functions of NAC and UBA domains in Peatl＇s thermal stability. [Method] Fusion expression vectors of Pearl protein and the 3 deletion mutants were constructed. The recombinant plasmids were induced by IPTG and the target proteins （Peatl, Peatl-△CD99,Peatl-△ND49 and Pearl-△ND108 ）were expressed obtained by AKTA and its thermal stability was analyzed. [Result] The research found that 3 deletion mutants have good thermal stability like Pearl. [Conclusion] The research demonstrated that the coexistence of NAC or UBA domains is not necessary to thermal stability of Pearl protein , and they may give the protein particular stability structure seperately.展开更多
The genome of the isolate of porcine reproductive and respiratory syndrome virus （PRRSV） from China, designated HPBEDV, was sequenced and analyzed. The size of the genome of HPBEDV was 15 334 nucleotides （nt）. Com...The genome of the isolate of porcine reproductive and respiratory syndrome virus （PRRSV） from China, designated HPBEDV, was sequenced and analyzed. The size of the genome of HPBEDV was 15 334 nucleotides （nt）. Comparative analysis of HPBEDV with the genomic sequences of the domestic and other isolates （JXA 1, HUN4, CH-1 a, B J-4, VR2332, and LV） revealed that HPBEDV shared 98.4, 98.0, 89.0, 88.7, and 88.6% identity with the American strain JXA1, HUN4, CH- 1a, BJ-4, and VR2332, respectively, but only 54.7% identity with the European reference strain Lelystad virus. The NSP2 gene had 2 850 nt and encoded 950 amino acids （aa）, with two discontiguous deletions of 1 aa and 29 aa at positions 482 and 534-562, respectively, relative to VR-2332. Also, phylogenetic analysis with the published PRRSV genomic sequences indicated that the newly emerging isolate form a clade with the VR-2332 isolates. Therefore, HPBEDV was a novel strain with deletions in NSP2 gene.展开更多
High-molecular-weight glutenin subunits（HMW-GSs） play a critical role in determining the viscoelastic properties of wheat dough. The HMW-GSs are encoded by Glu-A1, Glu-B1, and Glu-D1 loci on the long arms of chromos...High-molecular-weight glutenin subunits（HMW-GSs） play a critical role in determining the viscoelastic properties of wheat dough. The HMW-GSs are encoded by Glu-A1, Glu-B1, and Glu-D1 loci on the long arms of chromosomes 1A, 1B, and 1D, respectively. In the present study, four near-isogenic lines with different HMW-GS deletions and compositions at the Glu-A1 and Glu-D1 loci in Yangmai 18 background were used for quality analysis. Deletion in Glu-D1 showed much weaker gluten quality and dough strength than null Glu-A1 genotype and wild genotype（WT）, based on the measurements of sodium dodecyl sulfate（SDS）-sedimentation, lactic acid solvent retention capacity（SRC）, gluten index, development time, stability time, and alveograph P and L values. The deletion of Glu-D1 did not significantly affect grain hardness, grain protein content, water SRC, sodium carbonate SRC, and sucrose SRC. Double null genotype in Glu-A1 and Glu-D1 and single null genotype in Glu-D1 showed significantly higher cookie diameter, crispness, and lower cookie height compared with single null genotype in Glu-A1 and WT. These indicate that the null Glu-D1 genotype is useful for improvement of biscuit quality, and use of this germplasm would be a viable strategy to develop new wheat varieties for biscuit processing.展开更多
Under a given condition of crystallization, dark brown short rhombohedron crystals could be obtained from Δ nifZ MoFe protein purified from a nifZ deleted mutant strain of Azotobacter vinelandii Lipmann....Under a given condition of crystallization, dark brown short rhombohedron crystals could be obtained from Δ nifZ MoFe protein purified from a nifZ deleted mutant strain of Azotobacter vinelandii Lipmann. Systematic studies on the effect of concentrations of PEG 8000,MgCl 2, NaCl,Tris and buffer pH on the crystallization and crystal growth of the protein showed that the protein could not be crystallized in lower concentrations of the chemicals and lower buffer pH. A large amount of smaller crystals of the protein appeared in a week with gradual increasing in the chemical concentrations and pH≥8.0. When the chemical concentrations were further increased, the time for crystallization was increased and a few high grade crystals of larger size were formed. If the concentrations of the chemicals were continuously increased, many crystals with smaller size, and, sometimes of poor quality appeared again and eventually ceased to produce any crystals. The optimal concentration for each of the above mentioned chemicals varies with other variable factors. Only one bigger crystal (both of the longest two sides: 0.16 mm) could be obtained in a hanging drop of protein sample when the concentrations of PEG 8000, MgCl 2, NaCl,Tris and protein were kept at 1.86%, 300 mmol/L, 400 mmol/L, 53 mmol/L and 4.64 g/L , respectively, with Tris buffer pH 8.2.展开更多
Objective: To knock out the entire Luxs gene of Streptococcus mutans(S.mutans) UA159 strain via homologous recombination and construct a Luxs-deleted mutant strain of S. mutans. To study the difference between the aci...Objective: To knock out the entire Luxs gene of Streptococcus mutans(S.mutans) UA159 strain via homologous recombination and construct a Luxs-deleted mutant strain of S. mutans. To study the difference between the acid resistance of S. mutans Ingbritt C international standard strain and the acid resistance of LuxS mutant strain. Methods: Two DNA fragments locating in the upper and downstream of Luxs gene were amplified and a erythromycin resistance gene of PJT10 between them were engineered into PUC19 plasmid for constructing the recombination plasmid pUCluxKO. Electrotransformation of S.mutans cells with pUCluxKO-mutant resulted in isolation of erythromycin resistant S. mutans transformants, which was identified by polymerase chain reaction, V.harveyi BB170 luminescence bioassay and sequencing analysis. Solutions of S. mutans standard strain and LuxS mutant strain with same density were made and cultured at pH 3.5 to 7.0 BHI liquid for the same period.Terminal growth situation was compared.Firstly acidized in pH 5.5 BHI liquid,the two strains were cultured at pH 3.0 BHI liquid. The acid tolerance responses of the two strains were compared.Results:Restriction endonuclease analyses showed that pUCluxKO-mutant vector had been successfully recombined. The Luxs-deleted status of S.mutans mutants was confirmed by PCR with primers which were specific for the genes of Luxs and Erythromycin resistance. S.mutans mutant can not induce bioluminescence, indiating the mutant had been successfully recombined. After twenty generations of culture, the constructed Chinese S.mutans mutants were confirmed to be stable. Significant difference of aciduricity was observed between S.mutans standard strain and LuxS mutant strain.The acid resistance of standard strain was stronger than that of LuxS mutant strain.The two strains both displayed the capability of acid tolerance responses. Conclusion:The S.mutans gene allelic exchange plasmid is constructed correctively and a Luxs-negative mutants of S.mutans is constructed, which can help to further study the role of Luxs in the pathogenesis of S.mutans. LuxS mutant strain is more sensitive to acid inactivation,but the capability of acid tolerance responses exist still.展开更多
To identify differentially expressed genes from antigen-stimulated human dendritic cells (DC), subtractive cloning was adopted and more than ten novel genes differentially expressed were cloned. One is a deletion muta...To identify differentially expressed genes from antigen-stimulated human dendritic cells (DC), subtractive cloning was adopted and more than ten novel genes differentially expressed were cloned. One is a deletion mutant of heterogeneous nuclear ribonucleoprotein (hnRNP) M4 in which the residues from 159 to 197 of hnRNP M4 have been absent. The deletion mutant was shown to- be co-expressed with hnRNP M4 in cell lines. The mutant was expressed in antigen-stimulated DC but not in normal DC. Northern blot analysis revealed the presence of a major hnRNP M4 deletion mutant mRNA transcript of 2.4 kilobase with the highest levels in peripheral lymphocytes, lung, liver and spleen. It was also expressed in bone marrow-derived stromal cells (BMSC), BMSC treated with several cytokines but not in BMSC treated with TNF-a. The results revealed a new member of hnRNP family and suggested that hnRNP would participate in antigen process and presentation.展开更多
文摘The agp gene encoding the ADP-glucose pyrophosphorylase involved in cyanobacterial glycogen synthesis was amplified by PCR. The resulting agp fragment was cloned in plasmid pUC118 to generate plasmid pUCA. Part of the fragment within the agp DNA was deleted and replaced by an erythromycin resistance cassette to generate plasmid pUCAE, which was used to transform the Synechocystis sp. PCC 6803 wild-type strain and a mutant with resistance to erythromycin was obtained. PCR analysis of the genomic DNA from the resulting mutant indicated that the appropriate deletion and insertion indeed had occurred. The cell growth and Chl a, glycogen content in the mutant showed difference from those in the wild-type strain. The obtained biomass as well as the Chl a content in the mutant strain was higher than that of the wild-type strain, which suggested that the photosynthesis efficiency in the agp(-) strain was higher than that in the wild-type strain. No glycogen was found in the mutant, providing evidence for the correction of the mutant in physiological level.
基金Supported by the“973”Program(2003CB114204)the Science and Technology Plan(D0706005040431)~~
文摘[Objective] The research aimed to reveal the functions of NAC and UBA domains in Peatl＇s thermal stability. [Method] Fusion expression vectors of Pearl protein and the 3 deletion mutants were constructed. The recombinant plasmids were induced by IPTG and the target proteins （Peatl, Peatl-△CD99,Peatl-△ND49 and Pearl-△ND108 ）were expressed obtained by AKTA and its thermal stability was analyzed. [Result] The research found that 3 deletion mutants have good thermal stability like Pearl. [Conclusion] The research demonstrated that the coexistence of NAC or UBA domains is not necessary to thermal stability of Pearl protein , and they may give the protein particular stability structure seperately.
基金supported by the National Natural Sci-ence Foundation of China (30671563,30700597)the Natural Science Foundation of Gansu Province,China (0803RJZA050)
文摘The genome of the isolate of porcine reproductive and respiratory syndrome virus （PRRSV） from China, designated HPBEDV, was sequenced and analyzed. The size of the genome of HPBEDV was 15 334 nucleotides （nt）. Comparative analysis of HPBEDV with the genomic sequences of the domestic and other isolates （JXA 1, HUN4, CH-1 a, B J-4, VR2332, and LV） revealed that HPBEDV shared 98.4, 98.0, 89.0, 88.7, and 88.6% identity with the American strain JXA1, HUN4, CH- 1a, BJ-4, and VR2332, respectively, but only 54.7% identity with the European reference strain Lelystad virus. The NSP2 gene had 2 850 nt and encoded 950 amino acids （aa）, with two discontiguous deletions of 1 aa and 29 aa at positions 482 and 534-562, respectively, relative to VR-2332. Also, phylogenetic analysis with the published PRRSV genomic sequences indicated that the newly emerging isolate form a clade with the VR-2332 isolates. Therefore, HPBEDV was a novel strain with deletions in NSP2 gene.
基金supported by the Independent Innovation Funding for Agricultural Science and Technology of Jiangsu Province, China (CX(13)5070)the Natural Science Foundation of Jiangsu Province, China (BK20160448)the earmarked fund for China Agriculture Research System (CARS-03)
文摘High-molecular-weight glutenin subunits（HMW-GSs） play a critical role in determining the viscoelastic properties of wheat dough. The HMW-GSs are encoded by Glu-A1, Glu-B1, and Glu-D1 loci on the long arms of chromosomes 1A, 1B, and 1D, respectively. In the present study, four near-isogenic lines with different HMW-GS deletions and compositions at the Glu-A1 and Glu-D1 loci in Yangmai 18 background were used for quality analysis. Deletion in Glu-D1 showed much weaker gluten quality and dough strength than null Glu-A1 genotype and wild genotype（WT）, based on the measurements of sodium dodecyl sulfate（SDS）-sedimentation, lactic acid solvent retention capacity（SRC）, gluten index, development time, stability time, and alveograph P and L values. The deletion of Glu-D1 did not significantly affect grain hardness, grain protein content, water SRC, sodium carbonate SRC, and sucrose SRC. Double null genotype in Glu-A1 and Glu-D1 and single null genotype in Glu-D1 showed significantly higher cookie diameter, crispness, and lower cookie height compared with single null genotype in Glu-A1 and WT. These indicate that the null Glu-D1 genotype is useful for improvement of biscuit quality, and use of this germplasm would be a viable strategy to develop new wheat varieties for biscuit processing.
文摘Under a given condition of crystallization, dark brown short rhombohedron crystals could be obtained from Δ nifZ MoFe protein purified from a nifZ deleted mutant strain of Azotobacter vinelandii Lipmann. Systematic studies on the effect of concentrations of PEG 8000,MgCl 2, NaCl,Tris and buffer pH on the crystallization and crystal growth of the protein showed that the protein could not be crystallized in lower concentrations of the chemicals and lower buffer pH. A large amount of smaller crystals of the protein appeared in a week with gradual increasing in the chemical concentrations and pH≥8.0. When the chemical concentrations were further increased, the time for crystallization was increased and a few high grade crystals of larger size were formed. If the concentrations of the chemicals were continuously increased, many crystals with smaller size, and, sometimes of poor quality appeared again and eventually ceased to produce any crystals. The optimal concentration for each of the above mentioned chemicals varies with other variable factors. Only one bigger crystal (both of the longest two sides: 0.16 mm) could be obtained in a hanging drop of protein sample when the concentrations of PEG 8000, MgCl 2, NaCl,Tris and protein were kept at 1.86%, 300 mmol/L, 400 mmol/L, 53 mmol/L and 4.64 g/L , respectively, with Tris buffer pH 8.2.
文摘Objective: To knock out the entire Luxs gene of Streptococcus mutans(S.mutans) UA159 strain via homologous recombination and construct a Luxs-deleted mutant strain of S. mutans. To study the difference between the acid resistance of S. mutans Ingbritt C international standard strain and the acid resistance of LuxS mutant strain. Methods: Two DNA fragments locating in the upper and downstream of Luxs gene were amplified and a erythromycin resistance gene of PJT10 between them were engineered into PUC19 plasmid for constructing the recombination plasmid pUCluxKO. Electrotransformation of S.mutans cells with pUCluxKO-mutant resulted in isolation of erythromycin resistant S. mutans transformants, which was identified by polymerase chain reaction, V.harveyi BB170 luminescence bioassay and sequencing analysis. Solutions of S. mutans standard strain and LuxS mutant strain with same density were made and cultured at pH 3.5 to 7.0 BHI liquid for the same period.Terminal growth situation was compared.Firstly acidized in pH 5.5 BHI liquid,the two strains were cultured at pH 3.0 BHI liquid. The acid tolerance responses of the two strains were compared.Results:Restriction endonuclease analyses showed that pUCluxKO-mutant vector had been successfully recombined. The Luxs-deleted status of S.mutans mutants was confirmed by PCR with primers which were specific for the genes of Luxs and Erythromycin resistance. S.mutans mutant can not induce bioluminescence, indiating the mutant had been successfully recombined. After twenty generations of culture, the constructed Chinese S.mutans mutants were confirmed to be stable. Significant difference of aciduricity was observed between S.mutans standard strain and LuxS mutant strain.The acid resistance of standard strain was stronger than that of LuxS mutant strain.The two strains both displayed the capability of acid tolerance responses. Conclusion:The S.mutans gene allelic exchange plasmid is constructed correctively and a Luxs-negative mutants of S.mutans is constructed, which can help to further study the role of Luxs in the pathogenesis of S.mutans. LuxS mutant strain is more sensitive to acid inactivation,but the capability of acid tolerance responses exist still.
文摘To identify differentially expressed genes from antigen-stimulated human dendritic cells (DC), subtractive cloning was adopted and more than ten novel genes differentially expressed were cloned. One is a deletion mutant of heterogeneous nuclear ribonucleoprotein (hnRNP) M4 in which the residues from 159 to 197 of hnRNP M4 have been absent. The deletion mutant was shown to- be co-expressed with hnRNP M4 in cell lines. The mutant was expressed in antigen-stimulated DC but not in normal DC. Northern blot analysis revealed the presence of a major hnRNP M4 deletion mutant mRNA transcript of 2.4 kilobase with the highest levels in peripheral lymphocytes, lung, liver and spleen. It was also expressed in bone marrow-derived stromal cells (BMSC), BMSC treated with several cytokines but not in BMSC treated with TNF-a. The results revealed a new member of hnRNP family and suggested that hnRNP would participate in antigen process and presentation.
