Use of quantitative real-time and conventional PCR to assess the stability of the cp4 epsps transgene from Roundup Ready canola in the intestinal, ruminal, and fecal contents of sheep

The stability of transgenic DNA encoding the synthetic cp4 epsps protein in a diet containing Roundup Ready (RR) canola meal was determined in duodenal fluid (DF) batch cultures from sheep. A real-time TaqMan PCR assay was designed to quantify the degradation of cp4 epsps DNA during incubation in DF at pH 5 or 7. The copy number of cp4 epsps DNA in the diet declined more rapidly (P < 0.05) in DF at pH 5 as compared to pH 7. The decrease was attributed mainly to microbial activity at pH 7 and perhaps to plant endogenous enzymes at pH 5. The 62-bp fragment of cp4 epsps DNA detected by real-time PCR reached a maximum of approximately 1600 copies in the aqueous phase of DF at pH 7, whereas less than 20 copies were detected during incubations in DF at pH 5. A 1363-bp sequence of cp4 epsps DNA was never detected in the aqueous fraction of DF. Additionally, genomic DNA isolated from RR canola seed was used to test the persistence of fragments of free DNA in DF at pH 3.2, 5, and 7, as well as in ruminal fluid and feces. Primers spanning the cp4 epsps DNA coding region amplified sequences ranging in size from 300 to 1363 bp. Free transgenic DNA was least stable in DF at pH 7 where fragments less than 527 bp were detected for up to 2 min and fragments as large as 1363 bp were detected for 0.5 min. This study shows that digestion of plant material and release of transgenic DNA can occur in the ovine small intestine. However, free DNA is rapidly degraded at neutral pH in DF, thus reducing the likelihood that intact transgenic DNA would be available for absorption through the Peyer's Patches in the distal ileum.     

Efficacy of Organic Soil Amendments for Management of Heterodera glycines in Greenhouse Experiments

In a repeated greenhouse experiment, organic soil amendments were screened for effects on population density of soybean cyst nematode (SCN), Heterodera glycines, and soybean growth. Ten amendments at various rates were tested: fresh plant material of field pennycress, marigold, spring camelina, and Cuphea; condensed distiller’s solubles (CDS), ash of combusted CDS, ash of combusted turkey manure (TMA), marigold powder, canola meal, and pennycress seed powder. Soybeans were grown for 70 d in field soil with amendments and SCN eggs incorporated at planting. At 40 d after planting (DAP), many amendments reduced SCN egg population density, but some also reduced plant height. Cuphea plant at application rate of 2.9% (amendment:soil, w:w, same below), marigold plant at 2.9%, pennycress seed powder at 0.5%, canola meal at 1%, and CDS at 4.3% were effective against SCN with population reductions of 35.2%, 46.6%, 46.7%, 73.2%, and 73.3% compared with control, respectively. For Experiment 1 at 70 DAP, canola meal at 1% and pennycress seed powder at 0.5% reduced SCN population density 70% and 54%, respectively. CDS at 4.3%, ash of CDS at 0.2%, and TMA at 1% increased dry plant mass whereas CDS at 4.3% and pennycress seed powder at 0.1% reduced plant height. For Experiment 2 at 70 DAP, amendments did not affect SCN population nor plant growth. In summary, some amendments were effective for SCN management, but phytoxicity was a concern.     

Effect of processing on the recovery of recombinant beta-glucuronidase (rGUS) from transgenic canola

This study addresses the processing of transgenic canola seed for production of recombinant proteins by using beta-glucuronidase (rGUS) as a model protein. The major processing steps that were investigated included dry and wet grinding of the seed, solvent extraction of canola oil, and protein extraction. rGUS in canola seed was stable for at least 2 weeks of incubation at 38 degrees C and for more than 5 months at 10 degrees C. At 70 degrees C, the residual activity changed inversely to the initial moisture content of the seed. The comparison of wet and dry processing revealed no significant differences in protein recovery. rGUS was stable during the defatting of transgenic canola flakes with hexane at 66 degrees C, whereas 2-propanol extraction at the same temperature reduced the extractable enzyme activity by almost 50%. The particle size of the ground seed was important for the extraction efficiency. A faster extraction and greater protein yield was achieved by extracting particles with an average diameter equal to or smaller than 255 microm. More than 80% rGUS was extracted in one stage with sodium phosphate buffer of pH 7.5.     

Effects of expeller pressed camelina meal and/or canola meal on digestibility, performance and fatty acid composition of broiler chickens fed wheat-soybean meal-based diets

This experiment was conducted to compare the effects of graded levels of camelina meal and/or canola meal on digestibility, performance and fatty acid composition of broiler chickens. A total of 180-day-old male broiler chicks were randomly assigned to one of the six treatments. The control diet was based on wheat and soybean meal and contained 15% canola meal. The experimental diets contained 3%, 6%, 9%, 12% or 15% camelina meal added at the expense of canola meal. Chromic oxide (0.35%) was added to all diets as a digestibility marker. On the morning of day 22, birds were killed by cervical dislocation and their abdominal fat pad was obtained. The apparent total tract digestibility of dry matter and energy as well as nitrogen retention all declined linearly (p?相似文献   

Two seed coat-specific promoters are functionally conserved between <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> and <Emphasis Type="Italic">Brassica napus</Emphasis>

Canola is one of the most important cash crops in Canada, and a national project named “Designing Oilseeds for Tomorrow’s Market” was undertaken to improve seed meal quality of this strategically important crop. As a part of this project, our group is focusing on identifying seed coat-specific promoters for canola (Brassica napus). These promoters will be used to genetically modify canola seed coat to reduce or eliminate anti-nutritional components from the meal. The Arabidopsis thaliana BAN promoter (AtBANpro) and δVPE promoter (AtδVPEpro) were isolated and fused to GUS reporter gene to generate transgenic canola plants. These plants were analyzed by GUS staining and microtome sectioning which showed that both promoters are seed coat-specific in canola: AtBANpro in inner seed coat layer and AtδVPEpro in outer seed coat layer. Therefore, the two Arabidopsis promoters can be used to modify genes in seed coat of canola for further improving its seed qualities.     

Accumulation of a Brazil nut albumin in seeds of transgenic canola results in enhanced levels of seed protein methionine

We have increased the methionine content of the seed proteins of a commercial winter variety of canola by expressing a chimeric gene encoding a methionine-rich seed protein from Brazil nut in the seeds of transgenic plants. Transgenic canola seeds accumulate the heterologous methionine-rich protein at levels which range from 1.7% to 4.0% of the total seed protein and contain up to 33% more methionine. The precursor of the methionine-rich protein is processed correctly in the seeds, resulting in the appearance of the mature protein in the 2S protein fraction. The 2S methionine-rich protein accumulates in the transgenic seeds at the same time in development as the canola 11S seed proteins and disappears rapidly upon germination of the seed. The increase in methionine in the canola seed proteins should increase the value of canola meal which is used in animal feed formulations.     

Effects of untreated and heat-treated canola presscake on milk yield and composition of dairy cows

Five primiparous and five multiparous Holstein cows were used in two Latin square design experiments to determine effects of feeding unheated and heated canola presscake on milk yield and composition, and milk fatty acid concentrations of lactating dairy cows. Five diets that differed in level and source of dietary fat were formulated: a low fat control diet with 30 g kg⁻¹ fat from tallow, an unheated canola presscake supplemented diet (50 g kg⁻¹ fat), a heated canola presscake supplemented diet (50 g kg⁻¹ fat), a high tallow plus unheated canola meal supplemented diet (50 g kg⁻¹ fat), and a high tallow plus heated canola meal supplemented diet (50 g kg⁻¹ fat). In sacco ruminal degradability of heated and unheated canola presscake was compared with that of heated and unheated canola meal in a randomized complete block design using two ruminally fistulated cows. Heat treatment reduced ruminal DM and CP degradability of canola presscake. Multiparous cows fed diets supplemented with heated or unheated canola presscake produced more milk than those fed diets containing similar levels of fat from tallow with heated or unheated canola meal, respectively. High levels of fat from any diet reduced milk fat percentage for cows of either parity. Feeding heated canola products increased milk and milk protein yields in primiparous cows only, but cows of both parities fed diets containing canola presscake produced milk with lower concentrations of C12:0, C14:0, and C16:0 fatty acids than cows fed the canola meal and tallow diets, although concentrations of C18:1 n-9 were unaffected by fat source or level. Feeding canola products to dairy cows can alter milk fatty acid profile, but only primiparous cows have increased productivity as a result of feeding heated, versus unheated, canola presscake.     

A review of processing of feed ingredients to enhance diet digestibility in finfish

Replacement of fish meal with plant proteins in aquaculture diets presents several problems. Firstly, aquaculture diets, particularly diets for carnivorous fish species, are nutrient dense and may contain up to 450 g crude protein (CP)/kg. Such diets preclude the use of ingredients with only moderate CP content, such as pulses including peas and faba beans or oilseed meals including canola/rapeseed meal and flax. Secondly, virtually all crops contain heat-labile and heat-stable secondary compounds including protease inhibitors, tannins, lectins, phytate, dietary fibre and starch. Removal of heat-labile secondary compounds may be accomplished by extrusion or other heat treatment. However, elimination of heat-stable secondary compounds, and increasing the nutrient concentration of diets, requires fractionation of crops. Fractionation technologies range from low technology processes such as dehulling to medium technologies such as air classification to sophisticated technologies such as aqueous and solvent protein purification. Studies on the nutritional value of processed plant proteins in various fish species have consistently shown improved digestibility and growth compared to feeding unprocessed ingredients. This review examines effects of processing technologies on nutritional properties of soybean meal, canola meal, peas, lupins and flax in aquaculture diets.     

Aqueous extraction of beta-glucuronidase from transgenic canola: kinetics and microstructure

Aqueous extraction kinetics of recombinant beta-glucuronidase (rGUS) from transgenic canola (Brassica napus) was investigated in terms of the particle size and microstructural characteristics resulting from canola seed processing. The canola had been transformed to express recombinant GUS intracellulary in the seed, and electron microscopy showed that the cells are distributed among (1) disrupted cells in a thin layer at or adjacent to the particle surface, (2) disrupted cells within the interior, and (3) intact cells within the interior. A simple compartmental model containing two extractable pools and a third nonextractable pool fitted the batch extraction results very well. Comparing the rate constants from the model to estimates of expected transport rates from the observed cell fractions showed that the two extractable pools roughly correspond to the two disrupted cell fractions. Both flaking, causing more extensive cell wall damage throughout the seed, and grinding, increasing the total surface area, increase the size of the first pool and, therefore, the extraction yield. Mass transfer from the same type of pool from two types of processed seed behaved similarly. GUS extraction from the first extractable pool is 10-20 times faster (<1 min) than from the second extractable pool.     

Improvement of nutritive value of grass pea (Lathyrus sativus) seed meal in the formulated diets for rohu, Labeo rohita (Hamilton) fingerlings after fermentation with a fish gut bacterium

Eight isonitrogenous (35% crude protein approximately) and isocaloric (4.0 kcalg(-1) approximately) diets were formulated incorporating raw and fermented grass pea (Lathyrus sativus) seed meal at 10%, 20%, 30% and 40% levels by weight into a fish meal based diet and fed to rohu, Labeo rohita, fingerlings for 80 days and fish performance was studied. A particular bacterial strain (Bacillus sp.) isolated from the intestine of adult common carp (Cyprinus carpio) reared in the wild having significant amylolytic, cellulolytic, lipolytic and proteolytic activities were used for fermentation of seed meal for 15 days at 37 degrees C. Fermentation of grass pea seed meal was effective in significantly reducing the crude fibre content and anti-nutritional factors, such as tannins, phytic acid and the neurotoxin, beta-ODAP and enhancing the available free amino acids and fatty acids. In terms of growth response, feed conversion ratio and protein efficiency ratio, 30% fermented grass pea seed meal incorporated diet resulted in significantly (P < 0.05) better performance of rohu fingerlings. In general, growth and feed utilization efficiencies of fish fed diets containing fermented seed meal were superior to those fed diets containing raw seed meal. The apparent protein digestibility (APD) values decreased with increasing levels of raw seed meal in the diets. The APD for raw seed meal was lower at all levels of inclusion in comparison to those for the fermented seed meals. The highest deposition of carcass protein was recorded in fish fed the diet containing 40% fermented seed meal. The results indicated that fermented grass pea seed meal can be incorporated in carp diets up to 30% level compared to 10% level of raw seed meal.     

Phosphorus speciation in mature wheat and canola plants as affected by phosphorus supply

Background and aims

As plants approach maturity and start to senesce, the primary sink for phosphorus (P) is the seed but it is unclear how plant P status affects the resulting P concentration and speciation in the seed and remaining plant parts of the residues. This study was established to measure how P speciation in different parts of wheat and canola is affected by plant P status.

Methods

Wheat and canola grown in the glasshouse were supplied three different P rates (5, 30 and 60 kg P ha^?1 equivalent). At physiological maturity, plants were harvested and P speciation was determined for all plant parts (root, stem, leaf, chaff/pod and seed) and rates of P application, using solution ³¹P nuclear magnetic resonance (NMR) spectroscopy.

Results

Phytate was the dominant form of P in seed whereas orthophosphate was the dominant form of P in other plant parts. The distribution of P species varied with P status for canola but not for wheat. The phytate content of wheat chaff increased from 10 to 45 % of total P as the P rate increased. Canola pods did not show a similar trend, with most P present as orthophosphate.

Conclusions

Although minor differences were observed in P speciation across the three P application rates and plant parts, the effect of this on P cycling from residues into soil is likely to be relatively minor in comparison to the overall contribution of these residues to soil P pools. This glasshouse experiment shows the dominant P form in crop residues that is returned to soil after harvest is orthophosphate, regardless of plant P status.     

Reduction of phytic acid content in canola meal by Aspergillus ficuum in solid state fermentation process

Summary Solid state fermentation (SSF) of canola meal has been carried out to reduce its phytic acid content using Aspergillus ficuum NRRL 3135. In certain batches, a complete reduction of phytic acid content in canola meal was achieved in 48 h. A larger amount of biomass in the inoculum and older inoculum increased the rate of phytic acid hydrolysis. The optimum moisture content of the medium was found to be 67% for phytic acid hydrolysis in an SSF process. The substitution of water in the semi-solid medium with acetate buffer resulted in faster reduction of the phytic acid content. A 15% increase in the amount of protein after 120 h of incubation was observed in the treated meal. The crude phytase preparation extracted from the canola meal after it was treated in an SSF process was also used for reduction of the phytic acid content in new batches of canola meal both in semi-solid medium and in liquid medium. In the semi-solid medium, 58% of the phytic acid was hydrolysed at 45°C in 20 h, while 100% hydrolysis was recorded at 50°C in 12 h in the liquid medium. The SSF process seems to be beneficial for the upgrading of canola meal by reducing both its phytic acid content and increasing the amount of protein.Offprint requests to: Z. Duvnjak     

Plant products affect growth and digestive efficiency of cultured Florida pompano (Trachinotus carolinus) fed compounded diets

Costs of compounded diets containing fish meal as a primary protein source can be expected to rise as fish meal prices increase in response to static supply and growing demand. Alternatives to fish meal are needed to reduce production costs in many aquaculture enterprises. Some plant proteins are potential replacements for fish meal because of their amino acid composition, lower cost and wide availability. In this study, we measured utilization of soybean meal (SBM) and soy protein concentrate (SPC) by Florida pompano fed compounded diets, to determine the efficacy of these products as fish meal replacements. We also calculated apparent digestibility coefficients (ADCs) for canola meal (CM), corn gluten meal (CGM), and distillers dried grains with solubles (DDGS), following typical methods for digestibility trials. Juvenile Florida pompano were fed fish-meal-free diets containing graded levels of SBM and SPC, and weight gain was compared to a control diet that contained SBM, SPC, and fish meal. Fish fed diets that contained 25-30 percent SBM in combination with 43-39 percent SPC had weight gain equivalent to fish fed the control diet with fish meal, while weight gain of fish fed other soy combinations was significantly less than that of the control group. Apparent crude protein digestibility of CGM was significantly higher than that of DDGS but not significantly different from CM. Apparent energy digestibility of DDGS was significantly lower than CGM but significantly higher than CM. Findings suggested that composition of the reference diet used in a digestibility trial affects the values of calculated ADCs, in addition to the chemical and physical attributes of the test ingredient.     

Expression and purification of intact and functional soybean (Glycine max) seed ferritin complex in Escherichia coli

Soybean seed ferritin is essential for human iron supplementation and iron deficiency anemia prevention because it contains abundant bioavailable iron and is frequently consumed in the human diet. However, it is poorly understood in regards its several properties, such as iron mineralization, subunit assembly, and protein folding. To address these issues, we decided to prepare the soybean seed ferritin complex via a recombinant DNA approach. In this paper, we report a rapid and simple Escherichia coli expression system to produce the soybean seed ferritin complex. In this system, two subunits of soybean seed ferritin, H-2 and H-1, were encoded in a single plasmid, and optimal expression was achieved by additionally coexpressing a team of molecular chaperones, trigger factor and GroEL-GroES. The His-tagged ferritin complex was purified by Ni2+ affinity chromatography, and an intact ferritin complex was obtained following His-tagged enterokinase (His-EK) digestion. The purified ferritin complex synthesized in E. coli demonstrated some reported features of its native counterpart from soybean seed, including an apparent molecular weight, multimeric assembly, and iron uptake activity. We believe that the strategy described in this paper may be of general utility in producing other recombinant plant ferritins built up from two types of subunits.     

Nutritional evaluation of fermented black gram (Phaseolus mungo) seed meal in compound diets for rohu, Labeo rohita (Hamilton), fingerlings

Six isonitrogenous (approximately 35% crude protein) and isocaloric (approximately 4.0 kcal g⁻¹) diets were formulated incorporating raw and fermented black gram, Phaseolus mungo, seed meal at 20%, 30% and 40% levels by weight into a fishmeal‐based control diet fed to rohu, Labeo rohita, fingerlings (mean weight, 1.81 ± 0.21 g) for 80 days for a study of fish performance. A particular bacterial strain (Bacillus sp.) isolated from the intestine of adult common carp (Cyprinus carpio) reared in the wild having significant amylolytic, cellulolytic, lipolytic and proteolytic activities was used for fermentation of seed meal for 15 days at 37 ± 2°C. Fermentation of P. mungo seed meal was effective in significantly reducing the crude fibre content and antinutritional factors such as tannins and phytic acid, and enhancing available free amino acids and fatty acids. In terms of growth, feed conversion ratio and protein efficiency ratio, the 30% fermented black gram seed meal incorporated diet resulted in a significantly (P < 0.05) better performance of rohu fingerlings. In general, growth and feed utilization efficiencies of diets containing fermented seed meal were superior to diets containing raw seed meal. The apparent protein digestibility (APD) values decreased with increasing levels of raw seed meal in the diets. The APD for raw seed meal was lower at all levels of inclusion in comparison to those for the fermented seed meals. The maximum deposition of protein in the carcass was recorded in fish fed the diet containing 40% fermented seed meal. The results indicate that fermented black gram seed meal can be incorporated in carp diets up to the 30% level compared to the 10% level of raw seed meal.     

The utilization of canola and its constituents by lactating dairy cows

Three feeding trials and one nylon bag trial were conducted to determine the effect of supplementing a barley-based control diet with 3.5% canola oil (CO), 22% presscake (CPC) or 9% whole seed (WCS) on feed intake, digestibility, milk yield and composition of lactating dairy cows. Ruminal utilization of canola meal (CM), CPC and WCS was also determined. Increasing the level of fat in the diet had no significant effect on intake of concentrate or digestible energy, or on total tract digestibility of dry matter (DM), crude protein (CP) and acid detergent fibre. Addition of canola in the form of CPC and WCS gave greater energy and ether extract digestibility than C and CO (P < 0.05). Diet had no significant effect on milk production, yield of milk CP, milk lactose + ash, gross energetic efficiency of milk production, milk urea or minerals. Milk fat and 4% fat corrected milk (FCM) yield were similar with the C and CPC diets, and with the CO and WCS diets. But the CO and WCS diets gave less milk fat and FCM than the C diet (P < 0.05). Milk crude protein was higher (P < 0.05) on the WCS diet than on the C, CO and CPC diets, which were similar. Diets WCS, C and CO promoted similar levels of blood urea (BU) but BU levels with CPC and CO were lower than with the C diet (P < 0.05). Ruminal DM and CP disappearance of CM was lower than for WCS and CPC at all incubation times (P < 0.05).     

Phytase production and decrease of phytic acid content in canola meal byAspergillus carbonarius in solid-state fermentation

Solid-state fermentation (SSF) usingAspergillus carbonarius with canola meal as a substrate showed that production of phytase was associated with growth; maximum activity was achieved after 72 h. Apparent 25% and 10% increases in the protein content of the canola meal were noticed after 48 h and 72 h, respectively but total carbohydrate concentration had fallen by 25% by the end of fermentation. The rate of decrease of phytic acid content was optimum with a moisture content between 53% and 60%; homogenization of the inoculum for 120 s led to the greatest biomass and lowest phytic acid content. Inoculation of sterile meal led to lower phytic acid contents than inoculation of non-sterile meal.The authors are with the Department of Chemical Engineering, University of Ottawa, Ottawa, Ontario K1N 6N5, Canada     

Codon-modifications and an endoplasmic reticulum-targeting sequence additively enhance expression of an Aspergillus phytase gene in transgenic canola

Transgenic plants offer advantages for biomolecule production because plants can be grown on a large scale and the recombinant macromolecules can be easily harvested and extracted. We introduced an Aspergillus phytase gene into canola (Brassica napus) (line 9412 with low erucic acid and low glucosinolates) by Agrobacterium-mediated transformation. Phytase expression in transgenic plant was enhanced with a synthetic phytase gene according to the Brassica codon usage and an endoplasmic reticulum (ER) retention signal KDEL that confers an ER accumulation of the recombinant phytase. Secretion of the phytase to the extracellular fluid was also established by the use of the tobacco PR-S signal peptide. Phytase accumulation in mature seed accounted for 2.6% of the total soluble proteins. The enzyme can be glycosylated in the seeds of transgenic plants and retain a high stability during storage. These results suggest a commercial feasibility of producing a stable recombinant phytase in canola at a high level for animal feed supplement and for reducing phosphorus eutrophication problems.     

Seed specific expression and analysis of recombinant human adenosine deaminase (<Emphasis Type="Italic">hADA</Emphasis>) in three host plant species

The plant seed is a leading platform amongst plant-based storage systems for the production of recombinant proteins. In this study, we compared the activity of human adenosine deaminase (hADA) expressed in transgenic seeds of three different plant species: pea (Pisum sativum L.), Nicotiana benthamiana L. and tarwi (Lupinus mutabilis Sweet). All three species were transformed with the same expression vector containing the hADA gene driven by the seed-specific promoter LegA2 with an apoplast targeting pinII signal peptide. During the study, several independent transgenic lines were generated and screened from each plant species and only lines with a single copy of the gene of interest were used for hADA expression analysis. A stable transgenic canola line expressing the ADA protein, under the control of 35S constitutive promoter was used as both as a positive control and for comparative study with the seed specific promoter. Significant differences were detected in the expression of hADA. The highest activity of the hADA enzyme (Units/g seed) was reported in tarwi (4.26 U/g) followed by pea (3.23 U/g) and Nicotiana benthamiana (1.69 U/g). The expression of mouse ADA in canola was very low in both seed and leaf tissue compared to other host plants, confirming higher activity of seed specific promoter. Altogether, these results suggest that tarwi could be an excellent candidate for the production of valuable recombinant proteins.     

硼、镉元素及其交互作用与油菜产量和品质

硼和镉两种元素是影响油菜产量和品质的两个重要因素.硼是植物生长所必需的微量元素,施硼是油菜种植的必需环节;镉是植物生长的非必要元素,易在油菜体内富集,可能通过食物链危害人体健康.本文主要从镉含量与油菜食品安全品质角度考虑,阐述了油菜对镉的积累和耐受机制;同时,概括了前人总结的硼对油菜的产量和品质的影响.最后,结合本人研究区广西地区土壤有效硼含量低,全镉含量高的现状,提出运用硼镉交互作用机理,通过施加适量硼肥,提高油菜的产量和品质,消除土壤镉的潜在危害,从本质上改善该地区土壤存在的低硼高镉现状.