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排序方式: 共有125条查询结果,搜索用时 15 毫秒
21.
22.
Prevention of transgene flow from genetically modified crops to food crops and wild relatives is of concern in agricultural
biotechnology. We used genes derived from food crops to produce complete male sterility as a strategy for gene confinement
as well as to reduce the food purity concerns of consumers. Anther-specific promoters (A3, A6, A9, MS2, and MS5) were isolated from Brassica oleracea and B. rapa and fused to the β-glucuronidase (GUS) reporter gene and candidate genes for male sterility, including the cysteine proteases BoCysP1 and BoCP3, and negative regulatory components of phytohormonal responses involved in male development. These constructs were then introduced
into Arabidopsis thaliana. GUS analyses revealed that A3, A6, and A9 had tapetum-specific promoter activity from the anther meiocyte stage. Male sterility was confirmed in tested constructs
with protease or gibberellin insensitive (gai) genes. In particular, constructs with BoCysP1 driven by the A3 or A9 promoter most efficiently produced plants with complete male sterility. The tapetum and middle layer cells of anthers expressing
BoCysP1 were swollen and excessively vacuolated when observed in transverse section. This suggests that the ectopic expression of
cysteine protease in the meiocyte stage may inhibit programmed cell death. The gai gene also induced male sterility, although at a low frequency. This is the first report to show that plant cysteine proteases
and gai from food crops are available as a novel tool for the development of genetically engineered male-sterile plants.
Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. 相似文献
23.
Akimitsu Miyaji Masashi Suzuki Toshihide Baba Toshiaki Kamachi Ichiro Okura 《Journal of Molecular Catalysis .B, Enzymatic》2009,57(1-4):211-215
Particulate methane monooxygenase (pMMO), a copper-containing membrane protein, catalyzes methane hydroxylation under aerobic conditions. We found that the activity of pMMO was increased by catalase, implying that hydrogen peroxide (H2O2) is generated by pMMO with duroquinol, an electron donor for pMMO, and that the generated H2O2 inhibits pMMO activity. In addition, reversible inhibition of pMMO with H2O2 was observed upon treatment of pMMO with H2O2 followed by the addition of catalase, and H2O2 formation by pMMO with duroquinol was detected using a fluorescence probe. The redox behavior of type 2 copper in pMMO measured by the electron paramagnetic resonance revealed that H2O2 re-oxidizes the type 2 copper in pMMO reduced with duroquinol. 相似文献
24.
25.
Hiroshi Inoue Hiroyuki Kamachi Daigo Yamaya Miwa Oguma Munenori Noguchi 《Physiologia plantarum》2000,109(2):129-136
A proteolytic enzyme responsible for the breakdown of a 22-kDa protein, whose abundance decreases in thylakoid membranes during germination of green spores of the fern Osmunda japonica , was partially purified from the thylakoid membranes of quiescent spores by a combination of ammonium sulfate fractionation, ion-exchange chromatography on DEAE-Toyopearl 650 S and size-fractionation HPLC on G3000SW. The enzyme was found to be a cysteine endoproteinase, as judged by its dependency on sulfhydryl reagents and inhibition by E-64 and iodoacetate, and by the appearance of distinct proteolytic products that were not further degraded during prolonged reaction time. Highest protease activity was observed around pH 9.7, the activity being partially suppressed by cations. The Km of the 22-kDa protein as a substrate in the proteolysis was 67 μg ml−1 , equivalent to 3 μ M . The enzyme, with a native molecular mass of about 43 kDa, showed high specificity against the 22-kDa protein as a substrate. The isolated protease could not degrade the 22-kDa protein associated with fresh thylakoid membranes but digested the protein in the presence of 0.05% Triton X-100. 相似文献
26.
27.
Kozlowski PM Kamachi T Kumar M Yoshizawa K 《Journal of biological inorganic chemistry》2012,17(2):293-300
Density functional theory analysis was performed to elucidate the impact of one-electron reduction upon the initial step of
adenosylcobalamin-dependent enzymatic catalysis. The transition state (TS) corresponding to the Co–C bond cleavage and subsequent
hydrogen abstraction from the substrate was located. The intrinsic reaction coordinate calculations predicted that the reaction
consisting of Co–C5′ bond cleavage in [CoIII(corrin•)]–Rib (where Rib is ribosyl) and hydrogen-atom abstraction from the CH3–CH2–CHO substrate occurs in a concerted fashion. The computed activation energy barrier of the reaction (15.0 kcal/mol) was lowered
by approximately 54.5% in comparison with the reaction involving the positively charged cofactor model (Im–[CoIII(corrin)]–Rib+, where Im is imidazole; energy barrier = 33.0 kcal/mol). The Im base was detached during the TS search in the reaction involving
the one-electron-reduced analogue. Thus, to compare the energetics of the two reactions, the axial Im ligand detachment energy
for the Im–[CoIII(corrin•)]–Rib model was computed [7.6 kcal/mol (gas phase); 4.6 kcal/mol (water)]. Consequently, the effective activation energy
barrier for the reaction mediated by the Im-off [CoIII(corrin•)]–Rib was estimated to be 22.6 kcal/mol, which implied an overall 31.5% reduction in the energetic demands of the reaction.
Considering that the lengthened Co–Naxial bond has been observed in X-ray crystal structure studies of B12-dependent mutases, the catalytic impact induced by one-electron reduction of the cofactor is expected to be higher in the
presence of the enzymatic environment. 相似文献
28.
Fractionation of Nitrogen Isotopes by Glutamine Synthetase Isolated from Spinach Leaves 总被引:4,自引:0,他引:4
Yoneyama Tadakatsu; Kamachi Kazunari; Yamaya Tomoyuki; Mae Tadahiko 《Plant & cell physiology》1993,34(3):489-491
The isotopic fractionation of nitrogen in the reaction in vitroof glutamine synthetase isolated from spinach (Spinacia oleraceaL.) leaves was calculated from the changes in natural 15N abundance( 相似文献
29.
Purification, Characterization, and Immunological Properties of NADH-Dependent Glutamate Synthase from Rice Cell Cultures 总被引:1,自引:1,他引:0
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To obtain a monospecific antibody against NADH-dependent glutamate synthase (NADH-GOGAT; EC 1.4.1.14), the enzyme was purified to homogeneity from cultured rice cells (Oryza sativa) with column chromatography using Butyl Toyopearl 650M, Sephacryl S-300, Blue Sepharose CL-6B, and Butyl Toyopearl 650S. The specific activity at the final stage of the purification was 9.8 micromoles of glutamate formed per minute per milligram of protein. The yield was 6.1% and purification was 815-fold. Analysis by denaturing gel electrophoresis revealed a single polypeptide with an apparent molecular weight of 196,000, similar to the value of 194,000 estimated for the native protein. Apparent Km values for l-glutamine, 2-oxoglutarate, and NADH were 811, 76, and 3.0 micromolar, respectively. Neither NADPH nor l-asparagine substituted for NADH and l-glutamine, respectively. The enzyme had its absorption maxima at 273, 373, and 440 nanometers with a shoulder at 475 nanometers, suggesting that the rice NADH-GOGAT is a flavoprotein. Monospecific antibody raised against NADH-GOGAT purified from the rice cells was obtained as the first instance for the enzyme in higher plants. Immunological analyses showed that the antibody for rice cell NADH-GOGAT reacted with only the enzyme in extracts from the cells. The anti-NADH-GOGAT antibody did not recognize the ferredoxin-GOGAT purified from rice leaves, and likewise the anti-rice leaf ferredoxin-GOGAT antibody did not react with the NADH-GOGAT purified from the cultured rice cells. 相似文献
30.
Vascular bundle-specific localization of cytosolic glutamine synthetase in rice leaves 总被引:19,自引:4,他引:15
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Tissue localizations of cytosolic glutamine synthetase (GS1; EC 6.3.1.2), chloroplastic GS (GS2), and ferredoxin-dependent glutamate synthase (Fd-GOGAT; EC 1.4.7.1) in rice (Oryza sativa L.) leaf blades were investigated using a tissue-print immunoblot method with specific antibodies. The cross-sections of mature and senescent leaf blades from middle and basal regions were used for tissue printing. The anti-GS1 antibody, raised against a synthetic 17-residue peptide corresponding to the deduced N-terminal amino acid sequence of rice GS1, cross-reacted specifically with native GS1 protein, but not with GS2 after transfer onto a nitrocellulose membrane. Tissue-print immunoblots showed that the GS1 protein was located in large and small vascular bundles in all regions of the leaf blade prepared from either stage of maturity. On the other hand, GS2 and Fd-GOGAT proteins were mainly located in mesophyll cells. The intensity of the developed color on the membrane for GS1 was similar between the two leaf ages, whereas that for GS2 and Fd-GOGAT decreased during senescence. The tissue-specific localization of GS1 suggests that this GS isoform is important in the synthesis of glutamine, which is a major form of nitrogen exported from the senescing leaf in rice plants. 相似文献