Kunisuke Tanaka

Rice-based mucosal vaccine as a global strategy for cold-chain- and needle-free vaccination

Capable of inducing antigen-specific immune responses in both systemic and mucosal compartments without the use of syringe and needle, mucosal vaccination is considered ideal for the global control of infectious diseases. In this study, we developed a rice-based oral vaccine expressing cholera toxin B subunit (CTB) under the control of the endosperm-specific expression promoter 2.3-kb glutelin GluB-1 with codon usage optimization for expression in rice seed. An average of 30 μg of CTB per seed was stored in the protein bodies, which are storage organelles in rice. When mucosally fed, rice seeds expressing CTB were taken up by the M cells covering the Peyers patches and induced CTB-specific serum IgG and mucosal IgA antibodies with neutralizing activity. When expressed in rice, CTB was protected from pepsin digestion in vitro. Rice-expressed CTB also remained stable and thus maintained immunogenicity at room temperature for >1.5 years, meaning that antigen-specific mucosal immune responses were induced at much lower doses than were necessary with purified recombinant CTB. Because they require neither refrigeration (cold-chain management) nor a needle, these rice-based mucosal vaccines offer a highly practical and cost-effective strategy for orally vaccinating large populations against mucosal infections, including those that may result from an act of bioterrorism.

Gene structure and expression of rice seed allergenic proteins belonging to the α-amylase/trypsin inhibitor family

Genomic and two novel cDNA clones for rice seed allergenic protein (RA) belonging to the α-amylase/trypsin inhibitor family were isolated and their nucleotide sequences determined. Ten cysteine residues deduced from nucleotide sequences were completely conserved among three cDNA clones including a clone, RA17, reported previously. One genomic clone, λ4, contained two RA genes, RAG1 and RAG2. Although RAG1 was cloned at the 5′ portion only, two RA genes were arranged divergently. Nucleotide sequencing and DNA blotting analyses showed that RA are encoded by a multigene family consisting of at least four members. The transcriptional initiation site of RAG1 was localized at A, 26 bp upstream of the putative translational initiation codon, ATG, by the primer extension assay. The putative TATA box and CAAT box existed about 45 bp and 147 bp upstream of the transcription initiation site, respectively. A conserved sequence (ATGCAAAA) which was similar to the sequence (TGCAAAA) identified in rice glutelin promoters was observed in the 5′ region of the two genes. In addition, RNA blotting analyses provided that RA genes specifically expressed in ripening seed and their transcripts accumulated maximally between 15 and 20 days after flowering.

Cloning and sequencing of a cDNA encoding ascorbate peroxidase from Arabidopsis thaliana

A cDNA clone encoding ascorbate peroxidase (AP, EC 1.11.1.11) was isolated from a phage λgt11 library of cDNA fromArabidopsis thaliana by immunoscreening with monoclonal antibodies against the enzyme, and then sequenced. The cDNA insert hybridized to a 1.1 kb poly(A)+ RNA from leaves ofA thaliana. Genomic hybridization suggests that the cDNA obtained here corresponds to a single-copy gene. The N-terminal amino acid sequence ofArabidopsis AP was determined by protein sequencing of the immunochemically purified enzyme, and proved to be homologous to the N-terminal amino acid sequence of the chloroplastic AP of spinach. The predicted amino acid sequence of the mature AP ofA. thaliana, deduced from the nucleotide sequence, consists of 249 amino acid residues, which is 34% homologous with cytochromec peroxidase of yeast, but less homologous with other plant peroxidases. Amino acid residues at the active site of yeast cytochromec peroxidase are conserved in the amino acid sequence ofArabidopsis AP. The poly(dG-dT) sequence, which is a potential Z-DNA-forming sequence, was found in the 3′ untranslated region of the cDNA.

Mutants for rice storage proteins : 1. Screening of mutants for rice storage proteins of protein bodies in the starchy endosperm.

SummaryTo obtain genetic materials to breed qualitatively improved rice storage proteins, we screened about 3,000 mutant lines induced by the treatment of rice fertilized egg cell with N-methyl-N-nitrosourea (MNU). The screening was performed by comparing the profiles of sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) with that of the original variety, Kinmaze, especially focussing on the changes in polypeptides present in two kinds of protein bodies, PB-I and PB-II. We selected 17 mutant lines and classified them into 4 types on the basis of variations of the relative contents of the polypeptides. Determination of extracted protein in the starchy endosperm of the mutants revealed changes in the content of prolamin and glutelin but not globulin. In some mutants there was marked accumulation of 57 kDa polypeptide concomitant with the remarkable reduction of glutelin subunits. Treatment of the fertilized egg cell with MNU was found to be an effective method to induce mutations for storage proteins in protein bodies of rice.

Nucleotide sequence of a cDNA clone encoding a major allergenic protein in rice seeds Homology of the deduced amino acid sequence with members of α‐amylase/trypsin inhibitor family

A cDNA clone of rice major allergenic protein (RAP) was isolated from a cDNA library of maturing rice seeds. The cDNA had an open reading frame (486 nucleotides) which coded a 162 amino acid residue polypeptide comprising a 27‐residue signal peptide and a 135‐residue mature protein of Mr 14,764. The deduced amino acid sequence of RAP showed a considerable similarity to barley trypsin inhibitor [1983, J. Biol. Chem. 258, 7998–8003] and wheat α‐amylase inhibitor [1981, Phytochemistry 20, 1781–1784].

The Bean Seed Storage Protein [beta]-Phaseolin Is Synthesized, Processed, and Accumulated in the Vacuolar Type-II Protein Bodies of Transgenic Rice Endosperm.

The seed storage protein [beta]-phaseolin of the common bean (Phaseolus vulgaris L.) was expressed in the endosperm of transgenic rice (Oryza sativa L.) plants. The 5.1- or 1.8-kb promoter fragment of the rice seed storage protein glutelin Gt1 gene was fused transcriptionally to either the genomic or cDNA coding sequence of the [beta]-phaseolin gene. The highest quantity of phaseolin estimated by enzyme-linked immunosorbent assay was 4.0% of the total endosperm protein in the transgenic rice seeds. The phaseolin trait was segregated as a single dominant trait with a positive gene dosage effect and was stably inherited through three successive generations. Both phaseolin genomic and cDNA coding sequences were used to synthesize four isoforms of mature phaseolin protein with apparent molecular masses of 51, 48, 47, and 45 kD. Enzyme deglycosylation experiments indicated that the 51-kD form contains high-mannose N-glycans; the 48- and 47-kD forms have further modified N-glycans; and the 45-kD form is a nonglycosylated protein. Immunolabeling studies using light and electron microscopy demonstrated that phaseolin accumulates primarily in the vacuolar type-II protein bodies located at the periphery of the endosperm near the aleurone layer. We discuss the implications of these results on nutritional improvement of rice grains.

Three cDNA sequences coding for glutamine synthetase polypeptides in Oryza sativa L.

Atsushi Sakamoto i, Masahiro Ogawa 3, Takehiro Masumura l, Daisuke Shibata 1, Go Takeba 2, Kunisuke Tanaka 1 and Shoji Fujii 1 1Laboratory of Biochemistry, Faculty of Agriculture, Kyoto Prefectural University, Shimogamo, Kyoto 606, Japan; 2Laboratory of Applied Biology, Faculty of Living Science, Kyoto Prefectural University, Shimogamo, Kyoto 606, Japan; 3Laboratory of Food Production, The Research Institute for Food Science, Kyoto University, Ufi, Kyoto 611, Japan

Cloning and sequence analysis of a cDNA encoding rice glutaredoxin

A full‐length cDNA clone (RASC8) encoding glutaredoxin (thioltransferase) was isolated from a cDNA library of an aleurone layer prepared from a developing seed of rice (Oryza sativa L.). RASC8, 568bp in length, contained an ATG codon and two possible polyadenylation signals, and encoded 112 amino acid residues. Cys‐Pro‐Phe‐Cys, which is the active site and a highly conserved sequence among thioltransferases, was found in the deduced amino acid sequence. RASC8 was introduced into an expression vector pMALc2 and the translated product possessed thioltransferase activity.

FORMATION OF PHYTIC ACID IN CEREAL GRAINS

The ripening process of plants is characterized by the accumulation of substances in the seeds. The important mineral nutrient phosphorus is no exception to this accumulation process. The active transport of phosphorus to seeds from leaves and roots is an important part of the ripening. In the rice plant, at the end of the ripening, about 60% of the phosphorus in the whole plant is found in the grains.l Most of the phosphorus thus transported to the seeds exists in the form of phytic acid, inositol hexaphosphate. In cereal grains, 60-80% of the total phosphorus is found in phytic acid. It has been generally supposed that phytic acid functions as a storage form of phosphorus and is utilized as a source of phosphorus at germination. Recently, the occurrence of transphosphorylation between phytic acid and adenosine diphosphate has been suggested.2 Further, the occurrence of an enzyme catalyzing the transphosphorylation between phytic acid and guanosine diphosphate was established in mung bean.3 These findings show a more active participation of phytic acid as a phosphagen, rather than as a source of phosphorus at germination. We do not have much information regarding the formation of phytic acid, but the enzyme catalyzing the formation of inositol monophosphate has been fo~nd .4 -~ However, the phosphorylation mechanism in the biosynthesis of more highly phosphorylated inositol is unknown. Phytic acid is unique in having six phosphate ester linkages in one molecule of small molecular weight; hitherto, a highly phosphorylated compound such as phytic acid has not been known. From this point of view, it is supposed that phosphorylation of inositol is carried out through a mechanism different from the usual kinase reaction. A hypothesis for the mechanism of inositol phosphorylation, based on experiments on ripening grains of rice and wheat, is presented in this paper. According to this hypothesis, the phosphorylation of inositol in the formation of phytic acid does not occur in a sequential fashion through phosphorylated inositol, but occurs through a hypothetical phosphorylated inositol derivative. The occurrence of a new kind of polyphosphorylated inositol derivative in the ripening grains is postulated as a possible candidate.

cDNA cloning of an mRNA encoding a sulfur-rich 10 kDa prolamin polypeptide in rice seeds

Using a rice maturing seed pUC9 expression library, we isolated a cDNA clone corresponding to 10 kDa sulfurrich prolamin by immunoscreening. A longer cDNA clone was obtained from a λgtll library by plaque hybridization using this 32P-labeled cDNA as a probe. A polypeptide sequence composed of 134 amino acids was deduced from the nucleotide sequence. A 24 amino acid signal peptide was assigned by computer calculation for the membrane spanning region and Edman sequencing of the purified mature polypeptide. Remarkably, 20% of methionine and 10% of cysteine were found in the mature polypeptide as well as high contents of glutamine, and hydrophobic amino acids. Part of the amino acid sequence was homologous with a conserved cysteine-rich region found in other plant prolamins. Two repeats of amino acid sequence were found in the polypeptide.