Immunoproteomic and Two-Dimensional Difference Gel Electrophoresis Analysis of Arabidopsis Dehydration Response Element-Binding Protein 1A (DREB1A)-Transgenic Potato

Abstract

To increase the quality and yield of crops, transgenic plants have been developed that show enhanced resistance to insects or herbicides, or improved growth and nutrient status. In addition, transgenic plants expressing proteins associated with stress responses are being developed in an effort to produce crops with greater resistance to stresses such as cold, heat, and salt.1) Genetic engineering of a transcriptional factor may affect expressions of multiple genes, therefore, there may be unintended effects on protein expression levels in transgenic plants. For appropriate safety assessment, therefore, it is important to evaluate the proteomics of transgenic plants, particularly with respect to allergenicity.Even after the establishment of the 2003 Codex guidelines for methods to assess safety of foods derived from transgenic organisms, there is still controversy regarding methods used to test their allergenicity.2) Allergenicity of foods derived from transgenic organisms has been evaluated using immunological methods such as immunoblots, histamine release assays, skin prick tests, and oral exposure. 3,4) Immunoproteomic analysis of allergens with sera from allergic patients is a useful method for comprehensively comparing unknown allergens as well as known allergens in extracts of foods derived from transgenic organisms. Previously, we reported application of the immunoproteome to assess allergenicities of GH-1 (growth hormone 1)-transgenic amago salmon 5) and enhanced green fluorescent protein (EGFP)-transgenic chicken meat.6) The two-dimensional difference gel electrophoresis (2D-DIGE) method, a gel-based proteomic technology, is useful for evaluating relative differences in protein expression, if any, between non-transgenic and transgenic crops. The CyDye DIGE fluors used to label samples are spectrally resolvable, size-and charge-matched fluorescent dyes. 7,8) The method enables analysis of one or more samples on the same gel and is highly sensitive, and has a wide range of detection.Potato (Solanum tuberosum) is a staple food that is consumed worldwide. Various transgenic potatoes have been developed and their food safety has been assessed using serological and proteomic techniques.9-11) Several cases of potato allergies have been reported, 12-16) and a major potato allergen that causes hypersensitive reactions has been identified as patatin (Sola t 1).17) Potato allergy is considered to be cross-reactive with latex or pollens, and their cross-reactive fruit and vegetables. [18][19][20] Other potato allergens, cathepsin D-, cysteine-, and serine protease inhibitors, all of which are members of the trypsin inhibitor family, have been designated as Sola t 2, Sola t 3, and Sola t 4, respectively. 21) These allergens make up approximately 70-80% of total soluble potato proteins. 22)In this study, we investigated the allergenicity of stresstolerant transgenic potatoes 23,24) To produce crops that are more tolerant to stresses such as heat, cold, and salt, transgenic plants have been produced those express stre...

The Vitis vinifera C‐repeat binding protein 4 (VvCBF4) transcriptional factor enhances freezing tolerance in wine grape

“…, 1998). In another example, comparison of nontransgenic and transgenic potato expressing 35S:: AtDREB1A using 2D‐DIGE showed increased expression of only patatin, a major storage protein and decreased expression of lipoxygenase and starch synthase (Nakamura et al. , 2010).…”

“…However, such post-translational modifications are not always necessary as in the case of DREB1Arelated transcription factors (Liu et al, 1998). In another example, comparison of nontransgenic and transgenic potato expressing 35S::AtDREB1A using 2D-DIGE showed increased expression of only patatin, a major storage protein and decreased expression of lipoxygenase and starch synthase (Nakamura et al, 2010). Thus, there is precedent for ectopic expression of CBF-family transgenes having a relatively small effect on the proteome.…”

Salt and Drought Stress Tolerances in Transgenic Potatoes and Wild Species

“…No significant differences were observed on allergenicity, total glycoalkaloids, and its fractional component contents (Nakamura et al 2010).…”

Different Results of IgE Binding- and Crosslinking-Based Allergy Tests Caused by Allergen Immobilization

“…Considering that most of the allergens are soluble proteins, it seems important to evaluate the interaction of IgE and allergen in the liquid phase, as far as possible. In this context, similar caution should be exercised when applying the results of new techniques, such as multi-dimensional IgE blotting for allergenomics, and new allergen discovery, 17) such as allergen microarrays for high-throughput clinical diagnosis using basophils. 18) After immunochemical measurements like these, it seems that it is still necessary to confirm the ability of FcεRI crosslinking.…”