Expression of the β-subunit of β-conglycinin in seeds of transgenic plants

Soybean (Glycine max (L.) Merr.) seeds contain the storage protein -conglycinin, encoded by a multigene family. -Conglycinin consists of three subunits; , , and . A genomic clone for a -subunit of -conglycinin has been characterized by restriction-enzyme mapping and hybrid selected in-vitro translation followed by immunoprecipitation. In order to determine the developmental regulation of this -subunit gene, its expression was studied in seeds of transgenic petunia (Petunia hybrida) and tobacco (Nicotiana tabacum L.) plants. The -subunit expressed in seeds of petunia and tobacco was recognized by anti--conglycinin serum at a relative molecular mass of 53 000, equivalent to that of the native protein. Separation of the petunia-seed proteins by isoelectric focusing followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblot analysis showed that multiple isoelectric forms of the -subunit were produced. There was approximately a twofold variation in the accumulation of the -subunit protein in the mature seeds of transgenic petunia plants, each containing a single -subunit gene. However, the level of protein accumulation in mature seeds and the amount of -subunit mRNA in developing seeds was not correlated. Accumulation of the -subunit protein in transgenic seeds was less than the -subunit protein that accumulated in transgenic petunia seeds containing a single -subunit gene and less than the amount of the -subunit in mature soybean seeds which contain 8–13 -subunit genes. In transgenic tobacco plants, the accumulation of the -subunit protein in seeds was generally well correlated with the number of genes that were incorporated in the different transformants.Abbreviations kb kilobase - kDa kilodalton - M_r relative molecular mass - SDS-PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis     

Expression of a bacterial α-amylase gene in transgenic rice seeds

An alpha-amylase gene from Bacillus stearothermophilus under the control of the promoter of a major rice-seed storage protein was introduced into rice. The transgenic line with the highest alpha-amylase activity reached about 15,000 U/g of seeds (one unit is defined as the amount of enzyme that produces 1 mumol of reducing sugar in 1 min at 70 degrees C). The enzyme produced in the seeds had an optimum pH of 5.0-5.5 and optimum temperature of 60-70 degrees C. Without extraction or purification, the power of transgenic rice seeds was able to liquify 100 times its weight of corn powder in 2 h. Thus, the transgenic rice could be used for industrial starch liquefaction.     

Expression of the human milk protein β-casein in transgenic potato plants

A 1177 bp cDNA fragment encoding the human milk protein -casein was introduced into Solanum tuberosum cells under control of the auxin-inducible, bidirectional mannopine synthase mas12) promoters using Agrobacterium tumefaciens-mediated leaf disc transformation methods. Antibiotic-resistant plants were regenerated and transformants selected based on luciferase activity carried by the expression vector containing the human -casein cDNA. The presence of human -casein cDNA in the plant genome was detected by PCR and DNA hybridization experiments. Human -casein mRNA was identified in leaf tissues of transgenic plants by RT-PCR analysis. Human - casein was identified in auxin-induced leaf and tuber tissues of transformed potato plants by immunoprecipitation and immunoblot analysis. Human -casein produced in transgenic plants migrated in polyacrylamide gels as a single band with an approximate molecular mass of 30 kDa. Immunoblot experiments identified approximately 0.01% of the total soluble protein of transgenic potato leaf tissue as -casein. The above experiments demonstrate the expression of human milk - casein as part of an edible food plant. These findings open the way for reconstitution of human milk inedible plants for replacement of bovine milk in baby foods for general improvement of infant nutrition, and for prevention of gastric and intestinal diseases in children     

Extracellular targeting of the vacuolar tobacco proteins AP24, chitinase and β-1,3-glucanase in transgenic plants

The Nicotiana tabacum ap24 gene encoding a protein with antifungal activity toward Phytophthora infestans has been characterized. Analysis of cDNA clones revealed that at least three ap24-like genes are induced in tobacco upon infection with tobacco mosaic virus. Amino acid sequencing of the purified protein showed that AP24 is synthesized as a preproprotein from which an amino-terminal signal peptide and a carboxyl-terminal propeptide (CTPP) are cleaved off during post-translational processing. The functional role of the CTPP was investigated by expressing chimeric genes encoding either wild-type AP24 or a mutant protein lacking the CTPP. Plants expressing the wild-type construct resulted in proteins properly sorted to the vacuole. In contrast, the proteins produced in plants expressing the mutant construct were secreted extracellularly, indicating that the CTPP is necessary for targeting of AP24 to the vacuoles. Similar results were obtained for vacuolar chitinases and -1,3-glucanases of tobacco. The extracellularly targeted mutant proteins were shown to have retained their biological activity. Together, these results suggest that within all vacuolar pathogenesis-related proteins the targeting information resides in a short carboxyl-terminal propeptide which is removed during or after transport to the plant vacuole.     

Overexpression of a human α3/4-fucosyltransferase alters pollen development in transgenic tobacco plants

. In plants, the function of Ŏ-fucosylation remains largely unknown. To gain insight into the role of Ŏ-fucosylation during plant development, we generated transgenic tobacco plants overexpressing the human ō/4-fucosyltransferase (hFuc-TIII). Overexpressors clearly contained high amounts of hFuc-TIII and revealed a strong increase in !(1,4)fucosyltransferase activity in plant sexual organs. As a consequence, a more significant staining of Lewis^a motifs, the product of !(1,4)fucosyltransferase activity, was observed in transgenic pollen grains compared to those of controls. Here, we show that pollen grain development was altered in transgenic plants. The average size (polar and equatorial diameters) of mature pollen grains overexpressing hFuc-TIII was smaller than control pollen grains. Furthermore, whereas a reticulate cell wall surface was always observed on control pollen grains, a punctate and disorganized cell wall surface was observed on hFuc-TIII overexpressor pollen grains. In addition, transgenic pollen tube elongation was delayed compared to control pollen tube growth. This latter phenotype could at least explain the 35% reduction of seed production determined for the hFuc-TIII-overexpressing plants.     

Amino acid pair- and triplet-wise groupings in the interior of α-helical segments in proteins

A statistical approach has been applied to analyse primary structure patterns at inner positions of α-helices in proteins. A systematic survey was carried out in a recent sample of non-redundant proteins selected from the Protein Data Bank, which were used to analyse α-helix structures for amino acid pairing patterns. Only residues more than three positions apart from both termini of the α-helix were considered as inner. Amino acid pairings i, i+k (k=1, 2, 3, 4, 5), were analysed and the corresponding 20×20 matrices of relative global propensities were constructed. An analysis of (i, i+4, i+8) and (i, i+3, i+4) triplet patterns was also performed. These analysis yielded information on a series of amino acid patterns (pairings and triplets) showing either high or low preference for α-helical motifs and suggested a novel approach to protein alphabet reduction. In addition, it has been shown that the individual amino acid propensities are not enough to define the statistical distribution of these patterns. Global pair propensities also depend on the type of pattern, its composition and orientation in the protein sequence. The data presented should prove useful to obtain and refine useful predictive rules which can further the development and fine-tuning of protein structure prediction algorithms and tools.     

Expression of modified gene encoding functional human α-1-antitrypsin protein in transgenic tomato plants

Transgenic plants offer promising alternative for large scale, sustainable production of safe, functional, recombinant proteins of therapeutic and industrial importance. Here, we report the expression of biologically active human alpha-1-antitrypsin in transgenic tomato plants. The 1,182 bp cDNA sequence of human AAT was strategically designed, modified and synthesized to adopt codon usage pattern of dicot plants, elimination of mRNA destabilizing sequences and modifications around 5' and 3' flanking regions of the gene to achieve high-level regulated expression in dicot plants. The native signal peptide sequence was substituted with modified signal peptide sequence of tobacco (Nicotiana tabacum) pathogenesis related protein PR1a, sweet potato (Ipomoea batatas) sporamineA and with dicot-preferred native signal peptide sequence of AAT gene. A dicot preferred translation initiation context sequence, 38 bp alfalfa mosaic virus untranslated region were incorporated at 5' while an endoplasmic reticulum retention signal (KDEL) was incorporated at 3' end of the gene. The modified gene was synthesized by PCR based method using overlapping oligonucleotides. Tomato plants were genetically engineered by nuclear transformation with Agrobacterium tumefaciens harbouring three different constructs pPAK, pSAK and pNAK having modified AAT gene with different signal peptide sequences under the control of CaMV35S duplicated enhancer promoter. Promising transgenic plants expressing recombinant AAT protein upto 1.55% of total soluble leaf protein has been developed and characterized. Plant-expressed recombinant AAT protein with molecular mass of around approximately 50 kDa was biologically active, showing high specific activity and efficient inhibition of elastase activity. The enzymatic deglycosylation established proper glycosylation of the plant-expressed recombinant AAT protein in contrast to unglycosylated rAAT expressed in E. coli ( approximately 45 kDa). Our results demonstrate feasibility for high-level expression of biologically active, glycosylated human alpha-1-antitrypsin in transgenic tomato plants.     

Leaf developmental stage and tissue location affect the detection of β-glucuronidase in transgenic tobacco plants

Expression in Nicotiana tabaccum L. plants containing the -glucuronidase (GUS) gene under the control of the 35S (CaMV promoter) was affected by tissue type and ontogenic development of the leaves. GUS activity in ontogenetically younger leaves was 1003–1022 nmol 7-hydroxy-4-methylcoumarin (MU) formed mg^–1 (protein) min^–1 and in ontogenetically older leaves was only 140–198 nmol (MU) mg^–1 (protein) min^–1.     

Expression of Sambucus nigra agglutinin (SNA-I′) from elderberry bark in transgenic tobacco plants results in enhanced resistance to different insect species

Tobacco plants (Nicotiana tabacum cv Samsun NN) have been transformed with the gene encoding the type-2 ribosome-inactivating protein (RIP) SNA-I′ from elderberry (Sambucus nigra) under the control of the Cauliflower Mosaic Virus 35S promoter. Previous research confirmed that these plants synthesize, correctly process and assemble a fully active RIP. Variability in protein expression was observed within the transgenic lines. The effects of the type-2 RIP SNA-I′ delivered through a leaf feeding assay were evaluated in the laboratory on two economically important pest insects belonging to the orders of Hemiptera, the tobacco aphid (Myzus nicotianae) and Lepidoptera, the beet armyworm (Spodoptera exigua). In the experiment with aphids, significant effects were observed on the life parameters, such as survival, intrinsic rate of increase, net reproductive rate, mean generation time and mean daily offspring, whereas with caterpillars significant reduction in fresh weight as well as retardation in development were observed. In addition, significant increases in mortality were noted for insects fed on the transgenic lines as compared to wild type plants. This information provides further support for RIPs having a role in plant resistance to insect pest species.     

“Sink” regulation of photosynthetic metabolism in transgenic tobacco plants expressing yeast invertase in their cell wall involves a decrease of the Calvin-cycle enzymes and an increase of glycolytic enzymes

Leaves on transgenic tobacco plants expressing yeast-derived invertase in the apoplast develop clearly demarcated green and bleached sectors when they mature. The green areas contain low levels of soluble sugars and starch which are turned over on a daily basis, and have high rates of photosynthesis and low rates of respiration. The pale areas accumulate carbohydrate, photosynthesis is inhibited, and respiration increases. This provides a model system to investigate the sink regulation of photosynthetic metabolism by accumulating carbohydrate. The inhibition of photosynthesis is accompanied by a decrease of ribulose-1,5-bisphosphate and glycerate-3-phosphate, and an increase of triosephosphate and fructose-1,6-bisphosphate. The extracted activities of ribulose-1,5-bisphosphate carboxylase, fructose-1, 6-bisphosphatase and NADP-glyeraldehyde-3-phosphate dehydrogenase decreased. The activity of sucrose-phosphate synthase remained high or increased, an increased portion of the photosynthate was partitioned into soluble sugars rather than starch, and the pale areas showed few or no oscillations during transitions between darkness and saturating light in saturating CO₂. The increased rate of respiration was accompanied by an increased level of hexose-phosphates, triose-phosphates and fructose-1,6-bisphosphate while glycerate-3-phosphate and phosphoenolpyruvate decreased and pyruvate increased. The activities of pyruvate kinase, phosphofructokinase and pyrophosphate: fructose-6-phosphate phosphotransferase increased two- to four-fold. We conclude that an increased level of carbohydrate leads to a decreased level of Calvin-cycle enzymes and, thence, to an inhibition of photosynthesis. It also leads to an increased level of glycolytic enzymes and, thence, to a stimulation of respiration. These changes of enzymes are more important in middle- or long-term adjustments to high carbohydrate levels in the leaf than fine regulation due to depletion of inorganic phosphate or high levels of phosphorylated metabolites.Abbreviations Fru 1,6bisP fructose-1,6-bisphosphate - Fru 1,6bisPase fructose-1,6-bisphosphatase - Fru6P fructose-6-phosphate - Glc 1P glucose-1-phosphate - Glc6P glucose-6-phosphate - NADP-GAPDH NADP-dependent glyceraldehyde-3-phosphate dehydrogenase - PFK phosphofructokinase - PEP phosphoenolpyruvate - PFP pyrophosphate:fructose-6-phosphate phosphotransferase - PGA glycerate-3-phosphate - PK pyruvate kinase - Pi inorganic phosphate - Ru1,5bisP ribulose-1,5-bisphosphate - Rubisco ribulose-1,5-bisphosphate carboxylase-oxygenase - SPS sucrose-phosphate synthase - triose-P triose-phosphates     

Regulation of the α-expansin gene OsEXPA8 expression affects root system architecture in transgenic rice plants

Expansins are cell wall proteins implicated in the control of plant growth via loosening of the extracellular matrix, and are encoded by a large gene family. However, data linked to loss of function of single genes which support the role of expansins in root growth remain limited. In this study, we used RNA interference to examine the biological functions of the rice α-expansin gene OsEXPA8. Repression of OsEXPA8 expression in rice impaired the root system architecture and plant growth significantly, leading to shorter primary roots and fewer lateral roots. Accordingly, the cell size of the root vascular bundle reduced drastically. Notably, OsEXPA8 silencing impaired root hair elongation; moreover, plant height was clearly reduced. Transient expression of OsEXPA8-GFP in onion epidermal cells verified that OsEXPA8 is located on the cell wall. OsEXPA8 was expressed predominantly in the root and shoot of one-week-old rice seedlings, and highly induced by NaCl but suppressed by nitrate and phosphate starvation. In addition, atomic force microscopy was used to explore alterations in cell wall nanomechanics caused by OsEXPA8 protein reduction, which showed that the wall stiffness (Young’s modulus) of OsEXPA8-silenced suspension cells was increased significantly. Taken together, our results suggest that OsEXPA8 is critical for root system architecture, which supports the hypothesis that expansins are involved in enhancing plant growth by mediating cell wall loosening.     

Isolation of the promoter of a cotton β-galactosidase gene (GhGal1) and its expression in transgenic tobacco plants

β-galactosidases (GUS, EC 3.2.1.23) are character- ized by their ability to hydrolyze terminal, non-re- ducing β-D-galactosyl residues from β-D-galactosides and are widely distributed in microbes, plants and animals. To date, the primary structures of …     

Co-expression of α′ and β subunits of β-conglycinin in rice seeds and its effect on the accumulation behavior of the expressed proteins

A transgenic rice that produces both the α′ and β subunits of β-conglycinin has been developed through the crossing of two types of transgenic rice. Although the accumulation level of the α′ subunit in the α′β-transgenic rice was slightly lower than that in the transgenic rice producing only the α′ subunit, the accumulation level of the β subunit in the α′β-transgenic rice was about 60% higher than that in the transgenic rice producing only the β subunit. Results from sequential extraction and gel-filtration experiments indicated that part of the β subunit formed heterotrimers with the α′ subunit in a similar manner as in soybean seeds and that the heterotrimers interacted with glutelin via cysteine residues. These results imply that the accumulation level of the β subunit in the α′β-transgenic rice increases by an indirect interaction with glutelin. Immunoelectron microscopy revealed that the α′ and β subunits are localized in a low electron-dense region of protein body-II (PB-II) and that α′ homotrimers in the α′β-transgenic rice seeds seem to accumulate outside of this low electron-dense region.     

Expression of <Emphasis Type="Italic">Mucor circinelloides</Emphasis> gene for Δ6 desaturase results in the accumulation of high levels of γ-linolenic acid in transgenic tobacco

γ-Linolenic acid (GLA; C18:3 Δ6,9,12) is a nutritionally important fatty acid (FA) playing a vital role in biological structures and cellular functions, which is not produced in oil seed crops. Many oil seed plants, however, produce significant quantities of linoleic acid, a FA that could be converted into GLA by the enzyme Δ6 desaturase, if it is present. As a first step to produce GLA in oil seed crops, we isolated a cDNA encoding the Δ6-FA desaturase from filamentous fungus Mucor circinelloides M29. Expression of this gene in transgenic tobacco resulted in the accumulation of GLA to the levels of 23.1% of the total FA. The results suggested that it is feasible to introduce the M. circinelloides Δ6 desaturase gene into conventional oil crop to produce a large amount of GLA for functional foods and pharmaceutical products. This text was submitted by the authors in English. Y.L. Hao, X.H. Mei, and Y.B. Luo contributed equally to this work.