Image analysis of two-dimensional gel electrophoresis for comparative proteomics of transgenic and non-transgenic soybean seeds

Considering the importance of bidimensional electrophoresis and image analysis in comparative proteomics, the parameters that influence the analysis of protein expression of transgenic and non-transgenic soybean seeds were evaluated. The loaded mass of the proteins (150–500 µg), the pH separation range (3–10 or 4–7), and manual/automatic image editing were evaluated. Additionally, after optimizing the conditions, histograms and matchings were obtained in order to accurately analyze the variations (90%) in protein expression. From this, 10 proteins displayed significant differences in expression, and eight of them were characterized and identified by mass spectrometry.     

Improving metallomics information related to transgenic and non-transgenic soybean seeds using 2D-HPLC-ICP-MS and ESI-MS/MS

This work reports the use of 2D-HPLC-ICP-MS to enlarge metallomics information when considering soybean seeds. Separations using size exclusion chromatography (SEC) allowed the identification of three metal fractions: the first corresponding to molecular weights from 38.1 to 181.1 kDa, the second from 8.2 to 17.2 kDa and the third from 0.4 to 3.8 kDa. In a second dimension, using anion exchange chromatography (AEX), three sub-fractions containing Fe, Mg and Mn, one containing Cu, and three containing Co, Cu, Mg, Mn and Zn were obtained. After these separations, 33 proteins were identified using the ESI-MS/MS technique, and divided into four functional categories: plant growth/cell division, protein destination and storage, metabolism and unclassified proteins. Among the identified proteins, proteins previously related to metals were found.     

A transgenic,visual screenable marker for soybean seeds

Most soybean cultivars produce buff colored seeds due to a seed coat specific siRNA mechanism. This phenomenon is specifically limited to the seed coat and produces a strong visual effect, thus, a strategy to evade the silencing was used to produce a maternal transgenic marker for soybeans. Expression of a rice chalcone synthase transgene with little DNA sequence homology to the soybean siRNAs resulted in dark colored seed coats. This phenotype is the result of anthocyanin pigment production and does not appear to affect other tissues. This novel approach for producing an easily scored transgenic marker for soybean will facilitate high-throughput screening and analysis of transgenic soybean.     

Expression of functional recombinant human growth hormone in transgenic soybean seeds

We produced human growth hormone (hGH), a protein that stimulates growth and cell reproduction, in genetically engineered soybean [Glycine max (L.) Merrill] seeds. Utilising the alpha prime (α') subunit of β-conglycinin tissue-specific promoter from soybean and the α-Coixin signal peptide from Coix lacryma-jobi, we obtained transgenic soybean lines that expressed the mature form of hGH in their seeds. Expression levels of bioactive hGH up to 2.9% of the total soluble seed protein content (corresponding to approximately 9?g?kg(-1)) were measured in mature dry soybean seeds. The results of ultrastructural immunocytochemistry assays indicated that the recombinant hGH in seed cotyledonary cells was efficiently directed to protein storage vacuoles. Specific bioassays demonstrated that the hGH expressed in the soybean seeds was fully active. The recombinant hGH protein sequence was confirmed by mass spectrometry characterisation. These results demonstrate that the utilisation of tissue-specific regulatory sequences is an attractive and viable option for achieving high-yield production of recombinant proteins in stable transgenic soybean seeds.     

A sucrose binding protein homologue from soybean affects sucrose uptake in suspension-cultured transgenic tobacco cells

We have obtained Nicotiana tabacum transgenic cell lines expressing a sucrose binding protein (sbp) homologue gene from soybean (Glycine max L.), designated s-64, either in the sense or antisense orientation. Sense cell lines over-accumulated the S-64 protein, whereas the antisense cell lines had reduced levels of the endogenous homologue protein. Sucrose uptake experiments were conducted by incubating suspension-cultured tobacco cells with radiolabeled sucrose at pH 4.5 or 7.0. Raising the extracellular pH to 7.0 caused an inhibition of radiolabeled carbon uptake efficiency, which was attributed to the pH-sensitivity of cell-wall invertase (EC 3.2.1.26), H⁺/hexose transporter and/or H⁺/sucrose symporter activities. Because SBP-mediated sucrose uptake has been shown to be insensitive to extracellular pH in yeast, we performed the sucrose uptake experiments in sense and antisense cultured cells at pH 7.0. Under this condition, the level of SBP homologue correlated with the efficiency of radiolabeled uptake by the transgenic tobacco cells. Furthermore, manipulation of S-64 levels altered sucrose-cleaving activities in a metabolic compensatory manner. Enhanced accumulation of S-64 caused an increase in intracellular sucrose synthase (cleavage, EC 2.4.1.13) activity with a concomitant decline in cell-wall invertase activity. This result may reflect a metabolic adjustment of the sense cell lines caused by its high efficiency of direct sucrose uptake as disaccharide. In contrast, the level of cell-wall invertase activity was remarkably increased in antisense cells, favoring the invertase-dependent sugar uptake system. Collectively, these results may establish a functional link between radiolabeled influx and S-64 accumulation, suggesting that SBP affects sucrose uptake in suspension-cultured cells.     

Monitoring of Venus transgenic cell migration during pregnancy in non-transgenic rabbits

Cell transfer between mother and fetus were demonstrated previously in several species which possess haemochorial placenta (e.g. in humans, mice, rats, etc.). Here we report the assessment of fetal and maternal microchimerism in non-transgenic (non-TG) New Zealand white rabbits which were pregnant with transgenic (TG) fetuses and in non-TG newborns of TG does. The TG construct, including the Venus fluorophore cDNA driven by a ubiquitous cytomegalovirus enhancer, chicken ß-actin promoter (CAGGS), was previously integrated into the rabbit genome by Sleeping Beauty transposon system. Three different methods [fluorescence microscopy, flow cytometry and quantitative polymerase chain reaction (QPCR)] were employed to search for TG cells and gene products in blood and other tissues of non-TG rabbits. Venus positive peripheral blood mononuclear cells (PBMCs) were not detected in the blood of non-TG littermates or non-TG does by flow cytometry. Tissue samples (liver, kidney, skeletal and heart muscle) also proved to be Venus negative examined with fluorescence microscopy, while histology sections and PBMCs of TG rabbits showed robust Venus protein expression. In case of genomic DNA (gDNA) sourced from tissue samples of non-TG rabbits, CAGGS promoter-specific fragments could not be amplified by QPCR. Our data showed the lack of detectable cell transfer between TG and non-TG rabbits during gestation.     

A comparative study of water distribution and dehydrin protein localization in maturing pea seeds

In this study, the distribution of water in pea seeds after harvesting at different seed stages was traced by magnetic resonance imaging (MRI). MRI visualized the process of water loss in maturing pea seeds. MR images showed local inhomogeneities of water distribution inside seeds. The intensity of the signal coming from water declined from the inner to the outer part of cotyledon tissue. This spatial inhomogeneity of water signals inside cotyledons may be correlated with the gradient of storage substances accumulation within cotyledons. Tissue localization of dehydrins showed the presence of dehydrin protein in the area of protovascular tissue of both the embryo axis and cotyledons. The temporal accumulation of two dehydrin proteins with molecular masses of 30 and 35kDa correlated well with seed desiccation. The pattern of dehydrin localization reflected the pattern of water distribution in the protovascular bundles region of maturing pea embryos, suggesting the involvement of these proteins in promoting water influx into the vascular bundles.     

春秋花生种子细胞化学的比较研究

A comparative study was conducted on cytochemistry of spring and fall crop seeds in peanut cultivars Quanhua No. 10 and Shanyou 71 respectively. Lipids, protein, and polysaccharides in cells of axis and coteledon were simultaneously shown in the Epon812 buried section by means of cytochemistry, and their morphology, quantity and distribution were compared. Embryo cells of spring crop seed develop fully with big cell more vivid contrasting texture and more regularly disposed organelle, but the counterpart cells in fall crop seeds were not as much mature and their organelle arrangement appeared somewhat irregular. In cotyledon storage cells, there were also some difference between spring crop seed and fall crop seed. Cells of spring crop seeds were full of reserves, with more lipid and protein bodies that were closely ranged and extruded with each other. However, the cell structure in fall crop seeds was more loosely arranged, vacuoles had not been filled with protein, but starch grains accumulated more. Therefore, it was shown clearly that spring crop seeds have some advantages over fall crop seeds on production application. Moreover, some cytochemical techniques for demonstration of lipid, polysaccharide and protein in thick resin section and the stain protection were discussed in the paper.     

Comparative characterization of mesenchymal stem cells from eGFP transgenic and non-transgenic mice

Background  

Adipose derived- and bone marrow-derived murine mesenchymal stem cells (mMSCs) may be used to study stem cell properties in an in vivo setting for the purposes of evaluating therapeutic strategies that may have clinical applications in the future. If these cells are to be used for transplantation, the question arises of how to track the administered cells. One solution to this problem is to transplant cells with an easily identifiable genetic marker such as enhanced green fluorescent protein (eGFP). This protein is fluorescent and therefore does not require a chemical substrate for identification and can be visualized in living cells. This study seeks to characterize and compare adipose derived- and bone marrow-derived stem cells from C57Bl/6 mice and eGFP transgenic C57Bl/6 mice.     

A lipase-inhibiting protein from lipoxygenase-deficient soybean seeds

A lipase-inhibiting protein was isolated from lipoxygenase (LOX)-deficient soybean seeds. The molecular mass of the protein was 56.0-kDa and the N-terminal amino acid was blocked. The protein was identified by peptide mass fingerprinting in combination with matrix-assisted laser desorption ionization/time-of-flight mass spectrometry. The masses of the lysyl endopeptidase-digested peptides of the 56.0-kDa inhibiting protein were almost identical to the calculated masses of the theoretically predicted lysyl endopeptidase-treated peptides of beta-amylase from soybean seed. In a previous paper (Biosci. Biotechnol. Biochem., 62, 1498-1503, 1998), we reported that LOX-1, an isozyme of soybean seed LOX, inhibited hydrolysis of soybean oil by pancreatic lipase. Purified beta-amylase also inhibited lipase activity, although the magnitude of inhibition was weaker than that by LOX-1. Thus, there are at least two lipase-inhibiting proteins, one is a LOX and the other is a beta-amylase, in soybean seed.     

Embryo-specific expression of soybean oleosin altered oil body morphogenesis and increased lipid content in transgenic rice seeds

Oleosin is the most abundant protein in the oil bodies of plant seeds, playing an important role in regulating oil body formation and lipid accumulation. To investigate whether lipid accumulation in transgenic rice seeds depends on the expression level of oleosin, we introduced two soybean oleosin genes encoding 24 kDa proteins into rice under the control of an embryo-specific rice promoter REG-2. Overexpression of soybean oleosin in transgenic rice leads to an increase of seed lipid content up to 36.93 and 46.06 % higher than that of the non-transgenic control, respectively, while the overall fatty acid profiles of triacylglycerols remained unchanged. The overexpression of soybean oleosin in transgenic rice seeds resulted in more numerous and smaller oil bodies compared with wild type, suggesting that an inverse relationship exists between oil body size and the total oleosin level. The increase in lipid content is accompanied by a reduction in the accumulation of total seed protein. Our results suggest that it is possible to increase rice seed oil content for food use and for use as a low-cost feedstock for biodiesel by overexpressing oleosin in rice seeds.     

Evaluation of amino acid content and nutritional quality of transgenic soybean seeds with high-level tryptophan accumulation

Anthranilate synthase (AS) is a key regulatory enzyme in tryptophan (Trp) biosynthesis and is subject to feedback inhibition by Trp. The gene encoding a mutated feedback-resistant α subunit of rice AS (OASA1D) under the control of either a soybean glycinin gene promoter or the 35S promoter of cauliflower mosaic virus for seed-specific or constitutive expression, respectively, was introduced into soybean [Glycine max (L.) Merrill] by particle bombardment. A total of seven different transgenic lines that showed markedly increased accumulation of free Trp in their seeds were developed. The overproduction of free Trp was stably inherited in subsequent generations without any apparent detrimental effect on plant growth or reproduction. The total Trp content of transgenic seeds was also about twice that of nontransgenic seeds, whereas the amount of protein-bound Trp was not substantially affected by OASA1D expression. In spite of the marked increase in free Trp content, metabolic profiling by high-performance liquid chromatography coupled with mass spectrometry revealed little change in the amounts of other aromatic compounds in the transgenic seeds. We developed a rapid and feasible system based on farmed rainbow trout to evaluate the nutritional quality of a limited quantity of transgenic soybean seeds. Supplementation of fish food with OASA1D transgenic soybean seeds or with nontransgenic seeds plus crystalline Trp increased the growth rate of the farmed fish. These results indicate transformation with OASA1D is a reliable approach to improve the nutritional quality of soybean (or of other grain legumes) for human and animal food.     

Immunochemical study of changes in reserve proteins of germinating soybean seeds

Changes in the reserve proteins of soybean seeds (Glycine max) were investigated by the techniques of disc electrophoresis and disc immunoelectrophoresis. Three different antisera were used in these studies, an anti-whole soybean extract serum 129, an anti-11S soybean protein monospecific serum 102, and an anti-7S soybean protein monospecific serum 132. At least 6 antigenically distinct components were found to be present in the proteins of the isolated soybean protein bodies. These components are metabolized at different rates during germination. The major soybean protein (11S component) is found to be present even after 16 days of germination, whereas the 7S component disappears after the ninth day. Histochemical observations of cotyledon sections during germination are also reported.     

Effects of transgenic rootstocks on growth and development of non-transgenic scion cultivars in apple

Although cultivation of genetic modified (GM) annual crops has been steadily increasing in the recent 10 years, the commercial cultivation of GM fruit tree is still very limited and reports of field trials on GM fruit trees are rare. This is probably because development and evaluation of GM fruit trees require a long period of time due to long life cycles of trees. In this study, we report results from a field trial on three rolB transgenic dwarfing apple rootstocks of M26 and M9 together with non-transgenic controls grafted with five non-transgenic scion cultivars. We intended to investigate the effects of transgenic rootstock on non-transgenic scion cultivars under natural conditions as well as to evaluate the potential value of using the rolB gene to modify difficult-to-root rootstocks of fruit trees. The results showed that all rolB transgenic rootstocks significantly reduced vegetative growth including tree height regardless of scion cultivar, compared with the non-transgenic rootstocks. Flowering and fruiting were also decreased for cultivars grown on the transgenic rootstocks in most cases, but the fruit quality was not clearly affected by the transgenic rootstocks. Cutting experiment and RT-PCR analysis showed that the rolB gene was stably expressed under field conditions. PCR and RT-PCR analyses displayed that the rolB gene or its mRNA were not detectable in the scion cultivars, indicating no translocation of the transgene or its mRNA from rootstock to scion. Our results suggest that rolB modified rootstocks should be used in combination with vigorous scion cultivars in order to obtain sufficient vegetative growth and good yield. Alternatively, the rolB gene could be used to dwarf vigorous rootstocks of fruit trees or produce bonzai plants as it can significantly reduce the vegetative growth of plants.