[Objective] The aims were to construct a new suicide plasmid of Lactobacillus and gene deletion engineering bacteria of Lactobacillus with pUC19 vector. [Methods] pUC19-CM was constructed by inserting a chloramphenico...[Objective] The aims were to construct a new suicide plasmid of Lactobacillus and gene deletion engineering bacteria of Lactobacillus with pUC19 vector. [Methods] pUC19-CM was constructed by inserting a chloramphenicol resistant gene into the multi-cloning site of pUC19,and then two homologous fragments were cloned into each side of the pUC19-CM to construct suicide plasmid pUC19-CM-D. [Results] A replacement mutant strain,whose target gene was replaced by resistant gene,could be obtained by transforming the suicide plasmid pUC19-CM-D into Lactobacillus for resistance screening. [Conclusion] The construction and application of pUC19-CM-D provided a fast and efficient means of construction of gene deletion engineering bacteria of Lactobacillus,and laid a foundation for study of gene function of Lactobacillus.展开更多
Since the first terpenoid synthase cDNA was obtained by the reverse genetic approach from grand fir, great progress in the molecular genetics of terpenoid formation has been made with angiosperms and genes encoding a ...Since the first terpenoid synthase cDNA was obtained by the reverse genetic approach from grand fir, great progress in the molecular genetics of terpenoid formation has been made with angiosperms and genes encoding a monoterpene synthase, a sesquiterpene synthase, and a diterpene synthase. Tree killing bark beetles and their vectored fungal pathogens are the most destructive agents of conifer forests worldwide. Conifers defend against attack by the constitutive and inducible production of oleoresin that accumulates at the wound site to kill invaders and both flush and seal the injury. Although toxic to the bark beetle and fungal pathogen, oleoresin also plays a central role in the chemical ecology of these boring insects. Recent advances in the molecular genetics of terpenoid biosynthesis provide evidence for the evolutionary origins of oleoresin and permit consideration of genetic engineering strategies to improve conifer defenses as a component of modern forest biotechnology. This review described enzymes of resin biosynthesis, structural feathers of genes genomic intron and exon organization, pathway organization and evolution, resin production and accumulation, interactions between conifer and bark beetle, and engineering strategies to improve conifer defenses.展开更多
The difference of soluble sugar contents and starch synthetic key enzyme activities between high starch cultivar and low starch cultivar were studied in this experiment, of which FuXuan01, GR891, SC124, and SC201 were...The difference of soluble sugar contents and starch synthetic key enzyme activities between high starch cultivar and low starch cultivar were studied in this experiment, of which FuXuan01, GR891, SC124, and SC201 were used as materials. The results showed that the contents of reducing sugar in root tube of low starch cultivars were higher than those of high starch cultivars in all growth periods, the contents of sucrose in root tube of high starch cultivar were higher than those of low starch cultivars in the early growth period, but, lower in the late growth period, and there were almost no difference in the contents of soluble sugar in root tube between high starch cultivar and low starch cultivar in the early growth period, but notable difference in the late growth period and the soluble sugar contents of low starch cultivars were higher than those of high starch cultivars. It also-showed that the activities of ADPGPpase, SSS, and SBE （starch branching enzyme） of high starch cultivars were higher than those of low starch cultivars. It was evident that there were close correlations between the content of sucrose, reducing sugar and soluble sugar in root tube, and the activities of ADPGPpase, SSS, SBE, and the starch accumulation in the root tubers of cassava. It was quite evident in this experiment that the soluble sugar and starch synthetic key enzyme were main factors controlling starch accumulation in root tubers. These results provided important indication for physiological controlling in high starch cultivation and gene engineering breeding of high starch cultivar of cassava.展开更多
To obtain high efficiency of cleavage of thrombin in fusion protein containing a ANP fusetl to Re f pep-tides,the linker sequence deslgned as VIAGR which was dlfferent from GVRGPR formerly used was stud-ied. Plasmld p...To obtain high efficiency of cleavage of thrombin in fusion protein containing a ANP fusetl to Re f pep-tides,the linker sequence deslgned as VIAGR which was dlfferent from GVRGPR formerly used was stud-ied. Plasmld pHL carrying the fuslon gene Ref-NT-ANP downstream from PL Proruc)ter was derived fromexpression vector pLY1 by inserting the fragment of NT-ANP lnto lt. The exPresslon of fuslon gene waslnduced at 420,and the interested proteln Ref-NT-ANP accumlllated as inclusion bodies was lsolated bygradient centrifuge and then dissolved in 7 mol/L guanidinehydrochloride(Gdn-HCl). After dilution,renat-uration and dialyzation, the cleavage of thrombin was examined using samples with 1. 1 mol/1, (;dn-HC1and samples free of Gdn-HCl resI)ective1y. I)igestion result showed that the novel-ad()Pted cleavage sequencewas highly sensitive to thrombin when the substrate dissOlved in 1. 1 mol/I, tidn HCl. The time needed for87%cleavage (the ratio of substrate to thrombin was 5O pg/u)was less than 24 hOurs. This sequence described here which was sPecifically recognized by thrombin might be broadly applied in other fusion sys-tems.展开更多
Responses of 302 mitral/tufted (M/T) cells in the olfactory bulb were recorded from 42 anesthetized freely breathing rats using a 16-channel microwire electrode array.Saturated vapors of four pure chemicals,anisole,ca...Responses of 302 mitral/tufted (M/T) cells in the olfactory bulb were recorded from 42 anesthetized freely breathing rats using a 16-channel microwire electrode array.Saturated vapors of four pure chemicals,anisole,carvone,citral and isoamyl acetate were applied.After aligning spike trains to the initial phase of the inhalation after odor onset,the responses of M/T cells showed transient temporal features including excitatory and inhibitory patterns.Both odor-evoked patterns indicated that mammals recognize odors within a short respiration cycle after odor stimulus.Due to the small amount of information received from a single cell,we pooled results from all responsive M/T cells to study the ensemble activity.The firing rates of the cell ensembles were computed over 100 ms bins and population vectors were constructed.The high dimension vectors were condensed into three dimensions for visualization using principal component analysis.The trajectories of both excitatory and inhibitory cell ensembles displayed strong dynamics during odor stimulation.The distances among cluster centers were enlarged compared to those of the resting state.Thus,we presumed that pictures of odor information sent to higher brain regions were depicted and odor discrimination was completed within the first breathing cycle.展开更多
The aim of this study was to establish a quality-control method for calcineurin subunit B(CNB) biological activity determinations. CNB enhances the p-nitrophenylphosphate(p NPP) dephosphorylating activity of calcineur...The aim of this study was to establish a quality-control method for calcineurin subunit B(CNB) biological activity determinations. CNB enhances the p-nitrophenylphosphate(p NPP) dephosphorylating activity of calcineurin subunit A Δ316 mutant(CNAΔ316). A series of CNB concentrations were fitted to a four-parameter equation to calculate the corresponding p NPP maximum dephosphorylation rates. Values were calculated based on biological activity references using a parallel line method. The method was then validated for accuracy, precision, linearity, linear range, sensitivity, specificity, and robustness. The recovery results were greater than 98%. Intra-plate precision was 6.7%, with inter-plate precision of 10.8%. The coefficient of determination was greater than 0.98. The linear range was 0.05–50 μg m L?1, with sensitivity of 50 μg m L?1. Tested cytokines did not induce CNAΔ316 dephosphorylation of p NPP. The chosen CNAΔ316 concentration range did not affect activity determinations.展开更多
基金Supported by National Science &Technology Pillar Program in the Eleventh Five-year Plan Period (2007BAD75B06)Guangxi Sci-ence Foundation (0782003-4)~~
文摘[Objective] The aims were to construct a new suicide plasmid of Lactobacillus and gene deletion engineering bacteria of Lactobacillus with pUC19 vector. [Methods] pUC19-CM was constructed by inserting a chloramphenicol resistant gene into the multi-cloning site of pUC19,and then two homologous fragments were cloned into each side of the pUC19-CM to construct suicide plasmid pUC19-CM-D. [Results] A replacement mutant strain,whose target gene was replaced by resistant gene,could be obtained by transforming the suicide plasmid pUC19-CM-D into Lactobacillus for resistance screening. [Conclusion] The construction and application of pUC19-CM-D provided a fast and efficient means of construction of gene deletion engineering bacteria of Lactobacillus,and laid a foundation for study of gene function of Lactobacillus.
文摘Since the first terpenoid synthase cDNA was obtained by the reverse genetic approach from grand fir, great progress in the molecular genetics of terpenoid formation has been made with angiosperms and genes encoding a monoterpene synthase, a sesquiterpene synthase, and a diterpene synthase. Tree killing bark beetles and their vectored fungal pathogens are the most destructive agents of conifer forests worldwide. Conifers defend against attack by the constitutive and inducible production of oleoresin that accumulates at the wound site to kill invaders and both flush and seal the injury. Although toxic to the bark beetle and fungal pathogen, oleoresin also plays a central role in the chemical ecology of these boring insects. Recent advances in the molecular genetics of terpenoid biosynthesis provide evidence for the evolutionary origins of oleoresin and permit consideration of genetic engineering strategies to improve conifer defenses as a component of modern forest biotechnology. This review described enzymes of resin biosynthesis, structural feathers of genes genomic intron and exon organization, pathway organization and evolution, resin production and accumulation, interactions between conifer and bark beetle, and engineering strategies to improve conifer defenses.
文摘The difference of soluble sugar contents and starch synthetic key enzyme activities between high starch cultivar and low starch cultivar were studied in this experiment, of which FuXuan01, GR891, SC124, and SC201 were used as materials. The results showed that the contents of reducing sugar in root tube of low starch cultivars were higher than those of high starch cultivars in all growth periods, the contents of sucrose in root tube of high starch cultivar were higher than those of low starch cultivars in the early growth period, but, lower in the late growth period, and there were almost no difference in the contents of soluble sugar in root tube between high starch cultivar and low starch cultivar in the early growth period, but notable difference in the late growth period and the soluble sugar contents of low starch cultivars were higher than those of high starch cultivars. It also-showed that the activities of ADPGPpase, SSS, and SBE （starch branching enzyme） of high starch cultivars were higher than those of low starch cultivars. It was evident that there were close correlations between the content of sucrose, reducing sugar and soluble sugar in root tube, and the activities of ADPGPpase, SSS, SBE, and the starch accumulation in the root tubers of cassava. It was quite evident in this experiment that the soluble sugar and starch synthetic key enzyme were main factors controlling starch accumulation in root tubers. These results provided important indication for physiological controlling in high starch cultivation and gene engineering breeding of high starch cultivar of cassava.
文摘To obtain high efficiency of cleavage of thrombin in fusion protein containing a ANP fusetl to Re f pep-tides,the linker sequence deslgned as VIAGR which was dlfferent from GVRGPR formerly used was stud-ied. Plasmld pHL carrying the fuslon gene Ref-NT-ANP downstream from PL Proruc)ter was derived fromexpression vector pLY1 by inserting the fragment of NT-ANP lnto lt. The exPresslon of fuslon gene waslnduced at 420,and the interested proteln Ref-NT-ANP accumlllated as inclusion bodies was lsolated bygradient centrifuge and then dissolved in 7 mol/L guanidinehydrochloride(Gdn-HCl). After dilution,renat-uration and dialyzation, the cleavage of thrombin was examined using samples with 1. 1 mol/1, (;dn-HC1and samples free of Gdn-HCl resI)ective1y. I)igestion result showed that the novel-ad()Pted cleavage sequencewas highly sensitive to thrombin when the substrate dissOlved in 1. 1 mol/I, tidn HCl. The time needed for87%cleavage (the ratio of substrate to thrombin was 5O pg/u)was less than 24 hOurs. This sequence described here which was sPecifically recognized by thrombin might be broadly applied in other fusion sys-tems.
基金Project (Nos. 30970765 and 81027003) supported by the National Natural Science Foundation of China
文摘Responses of 302 mitral/tufted (M/T) cells in the olfactory bulb were recorded from 42 anesthetized freely breathing rats using a 16-channel microwire electrode array.Saturated vapors of four pure chemicals,anisole,carvone,citral and isoamyl acetate were applied.After aligning spike trains to the initial phase of the inhalation after odor onset,the responses of M/T cells showed transient temporal features including excitatory and inhibitory patterns.Both odor-evoked patterns indicated that mammals recognize odors within a short respiration cycle after odor stimulus.Due to the small amount of information received from a single cell,we pooled results from all responsive M/T cells to study the ensemble activity.The firing rates of the cell ensembles were computed over 100 ms bins and population vectors were constructed.The high dimension vectors were condensed into three dimensions for visualization using principal component analysis.The trajectories of both excitatory and inhibitory cell ensembles displayed strong dynamics during odor stimulation.The distances among cluster centers were enlarged compared to those of the resting state.Thus,we presumed that pictures of odor information sent to higher brain regions were depicted and odor discrimination was completed within the first breathing cycle.
基金supported by the National Important Novel Medicine Research Project (2012ZX09304010, 2013ZX09102062)the National Natural Science Foundation of China (31270849)
文摘The aim of this study was to establish a quality-control method for calcineurin subunit B(CNB) biological activity determinations. CNB enhances the p-nitrophenylphosphate(p NPP) dephosphorylating activity of calcineurin subunit A Δ316 mutant(CNAΔ316). A series of CNB concentrations were fitted to a four-parameter equation to calculate the corresponding p NPP maximum dephosphorylation rates. Values were calculated based on biological activity references using a parallel line method. The method was then validated for accuracy, precision, linearity, linear range, sensitivity, specificity, and robustness. The recovery results were greater than 98%. Intra-plate precision was 6.7%, with inter-plate precision of 10.8%. The coefficient of determination was greater than 0.98. The linear range was 0.05–50 μg m L?1, with sensitivity of 50 μg m L?1. Tested cytokines did not induce CNAΔ316 dephosphorylation of p NPP. The chosen CNAΔ316 concentration range did not affect activity determinations.
