Soh Hidaka

Production of waxy (amylose-free) wheats

The Waxy (Wx) protein has been identified as granule-bound starch synthase (GBSS; EC 24.1.21), which is involved in amylose synthesis in plants. Although common wheat (Triticum aestivum L.) has three Wx proteins, “partial waxy mutants” lacking one or two of the three proteins have been found. Using such partial waxy mutants, tetra- and hexaploid waxy mutants with endosperms that are stained red-brown by iodine were produced. Both mutants showed loss of Wx protein and amylose. This is the first demonstration of genetic modification of wheat starch.

Development of resistant transgenic soybeans with inverted repeat-coat protein genes of soybean dwarf virus

In an attempt to generate soybean plants resistant to soybean dwarf virus (SbDV), we transformed a construct containing inverted repeat-SbDV coat protein (CP) genes spaced by β-glucuronidase (GUS) sequences into soybean somatic embryos via microprojectile bombardment. Three T0 plants with an introduced CP gene were obtained, and one generated T1 seeds. The presence of the transgene in T1 plants was confirmed by PCR and Southern blot hybridization analysis, but expression of CP was not detected by northern blot hybridization analysis. Two months after inoculation of SbDV by aphid, T2 plants contained little SbDV-specific RNA and remained symptomless. These plants contained SbDV-CP-specific siRNA. These results suggest that the T2 plants achieved resistance to SbDV by an RNA-silencing-mediated process.

The waxy (Wx) proteins of maize, rice and barley

Abstract Maize waxy (Wx) protein, which has been detected as a single protein with a M r 60 000 by sodium dodecylsulphate polyacrylamide gel electrophoresis (SDS-PAGE), was separated into four isoforms by 2D-PAGE. The isoelectric point (p I ) of these isoforms ranged from 6.0 to 6.5. Starch isolation with SDS-buffer and heat-extraction of protein from starch granules had no effect on the 2D electrophoretic patterns of Wx protein. Rice and barley Wx proteins were also analysed by the same method and separated into four and three isoforms, respectively. Rice Wx protein isoforms had a M r of 60 000 as had been reported with a p I range from 6.5 to 7.2 while barley Wx protein isoforms had a M r of 61 000 and p I in the range of 5.8 to 6.3.

Complete nucleotide sequence of RNA 5 from cucumber mosaic virus (strain Y)

Cucumber mosaic virus Nucleotide sequence RNA 5

The promoter from the rice nuclear gene encoding chloroplast aldolase confers mesophyll-specific and light-regulated expression in transgenic tobacco

The rice genome contains at least four separate loci that encode aldolase isozymes. Among these, the aldolase P (AldP) gene, a nuclear gene coding for chloroplast aldolase, is expressed predominantly in the leaf blade mesophyll cells in rice. To dissect promoter elements that regulate such tissue- or cell type-specific expression, we constructed variousAldP promoter-β-glucuronidase (GUS) fusion genes and transferred them intoNicotiana tabacum (tobacco) plants. Analysis of GUS activities in the transgenic tobacco revealed the presence of at least two elements within 2.0 kbAldP promoter region. One is located within the segment from position − 2.0 kb to − 1.2 kb and acts as a negative element. The other is a positive element located between − 1.2 kb and − 0.31 kb that confers developmentally regulated, mesophyll cellspecific expression. In addition, the 1.2 kb rice promoter segment flanking the transcription start site contains an element(s) that serves as target for light induction in tobacco. The results suggest that theAIdP gene promoter of rice, a monocot promoter, can function in an essentially physiological manner in the dicot tobacco plant.

Soybean dwarf virus-resistant transgenic soybeans with the sense coat protein gene

We transformed a construct containing the sense coat protein (CP) gene of Soybean dwarf virus (SbDV) into soybean somatic embryos via microprojectile bombardment to acquire SbDV-resistant soybean plants. Six independent T0 plants were obtained. One of these transgenic lines was subjected to further extensive analysis. Three different insertion patterns of Southern blot hybridization analysis in T1 plants suggested that these insertions introduced in T0 plants were segregated from each other or co-inherited in T1 progenies. These insertions were classified into two types, which overexpressed SbDV-CP mRNA and accumulated SbDV-CP-specific short interfering RNA (siRNA), or repressed accumulation of SbDV-CP mRNA and siRNA by RNA analysis prior to SbDV inoculation. After inoculation of SbDV by the aphids, most T2 plants of this transgenic line remained symptomless, contained little SbDV-specific RNA by RNA dot-blot hybridization analysis and exhibited SbDV-CP-specific siRNA. We discuss here the possible mechanisms of the achieved resistance, including the RNA silencing.

Nucleotide sequence of the rice cytoplasmic aldolase cDNA

The cDNA coding for a cytoplasmic aldolase (cALD) of rice plant (Oryza sativa L. cv. Nihonbare) was cloned by screening from rice cDNA library with a cDNA probe (pMX71) specific to the maize cALD, kindly supplied by Freeling.M. (1). The sequence of two overlapping cDNA clones contained an open reading frame coding for a protein of 358 amino acids. Of the deduced amino acid sequence, 92% was identical with that of the maize cALD (2) by considering an insertion (residue 342-344) at the C-terminal region (residue 342 of the maize cALD).

Genomic structure of the rice aldolase isozyme C-1 gene and its regulation through a Ca2+-mediated protein kinase-phosphatase pathway

Complementary and genomic DNA clones coding for aldolase C-1, the fourth-type isozyme of aldolase in rice Oryza sativa L., have been characterized. The organization of the gene is quite similar to those encoding rice aldolase C-a and a maize cytoplasmic-type aldolase, in that introns are located in the same position. Amino acid sequences are highly conserved among cytoplasmic aldolases in plants. Expression of the gene in rice callus is activated by a protein phosphatase inhibitor okadaic acid, and is inhibited in the presence of thapsigargin, a reagent which increases calcium influx into the cytoplasm. The inhibition is rescued by the simultaneous addition of protein kinase inhibitor H-7. Thus, it is suggested that expression of the aldolase C-1 gene is regulated through a signal transduction pathway involving a Ca2+-mediated protein kinase-protein phosphatase system.

The application of the mutated acetolactate synthase gene from rice as the selectable marker gene in the production of transgenic soybeans

We investigated selective culturing conditions for the production of transgenic soybeans. In this culturing system, we used the acetolactate synthase (ALS)-inhibiting herbicide-resistance gene derived from rice (Os-mALS gene) as a selectable marker gene instead of that derived from bacteria, which interfered with the cultivation and practical usage of transgenic crops. T1 soybeans grown from one regenerated plant after selection of the ALS-targeting pyrimidinyl carboxy (PC) herbicide bispyribac-sodium (BS) exhibited herbicide resistance, and the introduction and expression of the Os-mALS gene were confirmed by genetic analysis. The selective culturing system promoted by BS herbicide, in which the Os-mALS gene was used as a selectable marker, was proved to be applicable to the production of transgenic soybeans, despite the appearance of escaped soybean plants that did not contain the Os-mALS transgene.

Comparison of plastid DNA replication in different cells and tissues of the rice plant.

In a previous study, we mapped replication origin regions of the plastid DNA around the 3′ end of the 23S rRNA gene in rice suspension-cultured cells. Here, we examined initiation of the plastid DNA replication in different rice cells by two-dimensional agarose gel electrophoresis. We show for the first time, to our knowledge, that the replication origin region of the plastid DNA differs among cultured cells, coleoptiles and mature leaves. In addition, digestion of the replication intermediates from the rice cultured cells with mung bean nuclease, a single-strand-specific nuclease, revealed that both two single strands of the double-stranded parental DNA were simultaneously replicated in the origin region. This was further confirmed by two-dimensional agarose gel analysis with single-stranded RNA probes. Thus, the mode of plastid DNA replication presented here differs from the unidirectional replication started by forming displacement loops (D-loops), in which the two D-loops on the opposite strands expand toward each other and only one parental strand serves as a template.