温度对菜籽蛋白质及植酸萃取率的影响

本文考察了pH=11.5和pH=12.0时,双液相萃取菜籽粕的蛋白质及植酸萃取率随温度变化的情况,提出pH=12.0,温度为40℃作为萃取菜籽蛋白的适宜条件。在该条件下,菜籽蛋白质的萃取率为71.5％,植酸率33.6％。     

High level expression in Escherichia coli,purification and properties of chloroplast fructose-1,6-bisphosphatase from rapeseed (Brassica napus) leaves

In chloroplasts, the light-modulated fructose-1,6-bisphosphatase catalyzes the formation of fructose 6-bisphosphate for the photosynthetic assimilation of CO₂ and the biosynthesis of starch. We report here the construction of a plasmid for the production of chloroplast fructose-1,6-bisphosphatase in a bacterial system and the subsequent purification to homogeneity of the genetically engineered enzyme. To this end, a DNA sequence that coded for chloroplast fructose-1,6-bisphosphatase of rapeseed (Brassica napus) leaves was successively amplified by PCR, ligated into the Ndel/EcoRI restriction site of the expression vector pET22b, and introduced into Escherichia coli cells. When gene expression was induced by isopropyl--d-thiogalactopyranoside, supernatants of cell lysates were extremely active in the hydrolysis of fructose 1,6-bisphosphate. Partitioning bacterial soluble proteins by ammonium sulfate followed by anion exchange chromatography yielded 10 mg of homogeneous enzyme per 1 of culture. Congruent with a preparation devoid of contaminating proteins, the Edman degradation evinced an unique N-terminal amino acid sequence [A-V-A-A-D-A-T-A-E-T-K-P-]. Gel filtration experiments and sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated that the (recombinant) rapeseed chloroplast fructose-1,6-bisphosphatases was a tetramer [160 kDa] comprised of four identical subunits. Like other chloroplast fructose-1,6-bisphosphatases, the recombinant enzyme was inactive at 1 mM fructose 1,6-bisphosphate and 1 mM Mg²⁺ but became fully active after an incubation in the presence of either 10 mM dithiothreitol or 1 mM dithiothreitol and chloroplast thioredoxin. However, at variance with counterparts isolated from higher plant leaves, the low activity observed in absence of reductants was not greatly enhanced by high concentrations of fructose 1,6-bisphosphate (3 mM) and Mg²⁺ (10 mM). In the catalytic process, all chloroplast fructose-1,6-bisphosphatases had identical features; viz., the requirement of Mg²⁺ as cofactor and the inhibition by Ca²⁺. Thus, the procedure described here should prove useful for the structural and kinetic analysis of rapeseed chloroplast fructose-1,6-bisphosphatase in view that this enzyme was not isolated from leaves.Abbreviation DTT dithiothreitol - PCR polymerase chain reaction - EDTA (ethylenedinitrilo)tetraacetic     

Effect of soil-applied NPK fertilizers on severity of black spot disease (Alternaria brassicae) and yield of oilseed rape

Effect of soil application of eight combinations of NPK fertilizers on the severity of black spot disease (BSD), caused by Alternaria brassicae (Sacc.) Berk., and yield of short duration oilseed rape (Brassica campestris L) were investigated under both pot and field conditions in 1987–88, 1988–89 and 1990–91. The severity of BSD was significantly greater (36–48%) on plants grown in ground treated with NP (N 90 kg ha^–1+P 40 kg ha^–1) applied as urea and single superphosphate respectively than on plants from the unfertilized control (NoPoKo) (o). However, the severity of BSD was significantly smaller (25–33%) when K (40 kg ha^–1) was applied as muriate of potash than in plants from control and NP treatments. The effect of NK (N 90 kg ha^–1+K 40 kg ha^–1) in decreasing the severity of BSD was increasingly more pronounced than the effects of PK (P 40 kg ha^–1+K 40 kg ha^–1), NP and K (40 kg ha^–1) applications. The decrease in the severity of BSD due to K was due to increased production in plants of phenolics which inhibited conidial germination and decreased sporulation of A. brassicae.The decrease in the severity of BSD due to NK application gave consistently increased seed yield 68% more than those of control and other treatments. The K-fertilized plants also showed increased resistance to lodging, increased 1000-seed weight and decreased seed infection. Seeds obtained from K-fertilized plants showed good seed germinability and vigorous seeding growth.     

The effect of 8 days of microgravity on regeneration of intact plants from protoplasts

The growth and development of protoplasts of rapeseed (Brassica napus L. cv Line) and carrot (Daucus carota L. cv. Navona) were studied onboard the Space Shuttle‘Discovery’during an 8-day International Microgravity Laboratory [IML-l) mission in January 1992. The Flight experiments were carried out in‘Biorack'. a fully controlled cell biological experimental facility. under microgravity conditions and in a l-g centrifuge. Parallel experiments were performed in a‘Biorack’module on the ground. After retrieval, some samples were subcultured on appropriate media and analysed for callus growth and regeneration to intact plants. The remainder were used for biochemical analysis. Samples fixed on board the Space Shuttle were kept in l% glutaraldehyde fixative at 4°C for 3–7 days for microscopy analysis after retrieval. Protoplasts exposed to microgravity conditions showed a delay in cell wall synthesis. Cells were swollen in appearance and formed cell aggregates with only few cells. Callus were obtained from protoplasts cultured under microgravity (Fogl). on the l-g centrifuge on board the shuttle (Flg), under normal l-g conditions on the ground (G1g) and on a centrifuge on the ground giving 1.4 g (Gl.4g). Regeneration of intact rapeseed plants was obtained from Flg. Glg and G1.4g. However, no plants were regenerated from protoplasts exposed to microgravity (Fog). Biochemical analysis indicated that the microgravity samples (Fog displayed a reduced packed cell volume, an increased concentration of soluble proteins per cell, and a reduced specific activity of peroxidase in the cytoplasm. Morphometric analysis of fixed samples demonstrated that 3-day old protoplasts under microgravity conditions were significantly larger than protoplasts kept on the l-g centrifuge in space. UItrastructural analysis by transmission electron microscopy showed that protoplasts exposed to microgravity conditions for 3 days had larger vacuoles and a slightly reduced starch content compared to Flg cells. Cell aggregates formed under microgravity conditions (Fog) had an average of 2–I cells per aggregate while aggregates formed under Flg had 8–12 cells.     

Valuable New Resistances Ensure Improved Management of Sclerotinia Stem Rot (Sclerotinia sclerotiorum) in Horticultural and Oilseed Brassica Species

Field resistances against Sclerotinia rot (SR) (Sclerotinia sclerotiorum) were determined in 52 Chinese genotypes of Brassica oleracea var. capitata, 14 Indian Brassica juncea genotypes carrying wild weedy Brassicaceae introgression(s) and four carrying B‐genome introgression, 22 Australian commercial Brassica napus varieties, and 12 B. napus and B. juncea genotypes of known resistance. All plants were individually inoculated by securing an agar disc from a culture of S. sclerotiorum growing on a glucose‐rich medium to the stem above the second internode with Parafilm tape. Mean stem lesion length across tested genotypes ranged from <1 to >68 mm. While there was considerable diversity within the germplasm sets from each country, overall, 65% of the B. oleracea var. capitata genotypes from China showed the highest levels of stem resistance, a level comparable with the highest resistance ever recorded for oilseed B. napus or B. juncea from China or Australia. One Indian B. juncea line carrying weedy introgression displayed a significant level of both stem and leaf resistance. However, the vast majority of commercial Australian oilseed B. napus varieties fell within the most susceptible 40% of genotypes tested for stem disease. There was no correlation between expressions of stem versus leaf resistance, suggesting their independent inheritance. A few Chinese B. oleracea var. capitata genotypes that expressed combined extremely high‐level stem (≤1 mm length) and leaf (≤0.5 mean number of infections/plant) resistance will be particularly significant for developing new SR‐resistant horticultural and oilseed Brassica varieties.     

Transgenic rapeseed plants obtained by the microinjection of DNA into microspore-derived embryoids

Summary A novel method in the field of genetic engineering of higher plants is presented: microinjection into multicellular structures which have a high competence for plant regeneration through embryogenesis. Microspore-derived embryoids of Brassica napus L. were individually selected and microinjected with NPT II gene constructions. High frequency regeneration of haploid plants through embryogenesis was achieved within 8 weeks. Transformation efficiencies between 27% and 51% were determined by DNA dot blot analysis of primary regenerants. Stable integration of fulllength microinjected genes into high molecular weight DNA was proven by Southern analysis of genomic DNA isolated from regenerated plants. Transformed plants were tested for expression of the NPT II gene by enzyme assay. The chimeric nature of the primary regenerants was demonstrated after their in vitro segregation through secondary embryogenesis into pure transformants.     

Modification of the gravitropic response of seedling roots of raneseed (Brassica napus) transformed by Agrobacterium rhizogenes A4

We have examined the growth and gravitropic response of seedling roots of rapeseed ( Brassica napus . CrGC5–1) transformed by Agrobacterium rhizogenes A4, in order to evaluate if this could constitute a new model system for the study of gravitropism. The transformed clone chosen for study had integrated full-length TL- and TR-DNA from pRi (the root inducing plasmid), and thus included all of the agrobacterial genes potentially involved in the modified phenotype of transformed plants. In the vertical position, the growth rate of transformed roots was higher than controls. During 24 h of continuous stimulation, the optimal angle for gravitropic bending in normal roots was 135° (with respect to the gravity axis), with decreasing response at 90° and 45°. For transformed roots, slight curvature developed at 45° and at 90°, and stronger curvature was observed at 135°, though transformed roots tips never reached the vertical position. The minimum stimulation time necessary to elicit a response (presentation time) was also determined: it was signficantly shorter in normal roots (80 s) than in transformed ones (120 s). The results show that pRi transformed roots are less sensitive to gravity than normal roots.     

Temperature controls both gametophytic and sporophytic development in microspore cultures of Brassica napus

Summary Temperature controls the developmental fate of isolated Brassica napus microspores in vitro. Culture at 32.5°C leads to sporophytic development and the formation of embryos. Here we show that culture at 17.5°C leads to gametophytic development, and the formation of pollen-like structures at high frequencies (up to 80% after 7 days in culture). Early stages of both developmental pathways are observed in culture at 25.0°C, and embryos are produced at low frequencies (0.7%) at that temperature. Culturing B. napus microspores at 32.5°C versus 17.5°C brings the switch from gametophytic to sporophytic development under simple experimental control and provides a convenient tool for investigating the cellular and molecular mechanisms controlling this developmental switch.     

The impact of reducing fatty acid desaturation on the composition and thermal stability of rapeseed oil

Oilseed rape (Brassica napus) is the third largest source of vegetable oil globally. In addition to food uses, there are industrial applications that exploit the ability of the species to accumulate the very‐long‐chain fatty acid (VLCFA) erucic acid in its seed oil, controlled by orthologues of FATTY ACID ELONGASE 1 (Bna.FAE1.A8 and Bna.FAE1.C3). The proportion of polyunsaturated fatty acids (PUFAs) in rapeseed oil is predicted to affect its thermal stability and is controlled by orthologues of FATTY ACID DESATURASE 2, particularly Bna.FAD2.C5. Our aim was to develop rapeseed lines combining high erucic and low PUFA characters and to assess the impact on thermal stability of the oil they produce. The new type of rapeseed oil (high erucic low polyunsaturate; HELP) contained a substantially greater proportion of erucic acid (54%) compared with high erucic rapeseed oil (46%). Although the total VLCFA content was greater in oil from HELP lines (64%) than from high erucic rapeseed (57%), analysis of triacylglycerol composition showed negligible incorporation of VLCFAs into the sn‐2 position. Rancimat analysis showed that the thermal stability of rapeseed oil was improved greatly as a consequence of reduction of PUFA content, from 3.8 and 4.2 h in conventional low erucic and high erucic rapeseed oils, respectively, to 11.3 and 16.4 h in high oleic low PUFA (HOLP) and HELP oils, respectively. Our results demonstrate that engineering of the lipid biosynthetic pathway of rapeseed, using traditional approaches, enables the production of renewable industrial oils with novel composition and properties.