Yoshiki Habu

Transgenic rice plants expressing a trypsin inhibitor are resistant against rice stem borers, Chilo suppressalis.

A synthetic gene, mwti1b, coding for a winged bean trypsin inhibitor WTI‐1B, has been introduced and expressed in rice plants, Oryza sativa. Protein extracts from transgenic rice plants expressing the trypsin inhibitor inhibited the gut proteases of larvae of the serious insect pest, the rice stem borer, Chilo suppressalis (Lepidoptera: Pyralidae) in vitro. The growth of larvae reared on transgenic rice plants expressing WTI‐1B at more than 1 ng/10 μg total protein was significantly retarded compared to that on non‐transgenic control plants.

Floricultural Traits and Transposable Elements in the Japanese and Common Morning Gloriesaa

ABSTRACT: The Japanese morning glory has an extensive history of genetic studies and over 200 different spontaneous mutant lines have been described. Of these, we identified that two mutable alleles, flecked and speckled, for flower variegations are caused by integration of transposable elements, belonging to the En/Spm family, into the DFR‐B and CHI genes for flower pigmentation, respectively. The mutable flaked allele of the common morning glory bearing variegated flowers is caused by insertion of a new transposable element, Tip 100, into one of the CHS genes for pigmentation and that Tip 100 belongs to the Ac/Ds family. These results are discussed with regard to spontaneous transposon mutagenesis and generation of floricultural traits of morning glories.

Direct isolation of differentially expressed genes from a specific chromosome region of common wheat: application of the amplified fragment length polymorphism-based mRNA fingerprinting (AMF) method in combination with a deletion line of wheat.

Abstract The amplified restriction fragment length polymorphism (AFLP)-based mRNA fingerprinting (AMF) method makes it possible systematically and conveniently to identify differentially expressed cDNAs with high reproducibility. We have applied the AMF method to the cloning of the Q gene of common wheat, which is located on the long arm of chromosome 5A and pleiotropically controls the spike morphology and the threshing character of seeds. Using the AMF method, we compared the fingerprints of mRNA samples extracted from the young spikes of Triticum aestivum cv. Chinese Spring (CS) carrying the Q gene to those of a chromosome deletion line of CS, namely, q5, which lacks 15% of 5AL including the Q gene. Approximately 12,200 fragments were produced after PCR with 256 primer combinations. Of these, 92 fragments were differentially expressed between CS and q5. Northern and Southern analyses showed that 16 fragments gave specific or relatively stronger transcript signals in CS, and these clones were present in single copy or in low copy numbers in the wheat genome. Four clones were genetically mapped to the region deleted in q5. Subsequently, one clone, pTaQ22, was mapped at the same locus as the Q gene, indicating that pTaQ22 corresponds to the Q gene or is tightly linked to it. DNA sequence data showed that pTaQ22 had no homology to any known genes, thus suggesting a novel function for this gene in flower morphogenesis. This AMF method might provide a straightforward method for isolating genes in the hexaploid background of common wheat.

Alteration of methylation profiles in distinct cell lineages of the layers during vegetative propagation in carnations (Dianthus caryophyllus)

In vegetatively propagated plants, branches of periclinal chimera give rise to bud mutants, or “sports”, which have been used to breed new cultivars. Here, we have examined DNA methylation profiles in the cells of the L1 and L2+3 layers from single plants of vegetatively propagated carnations. The band patterns of methylation-sensitive amplified fragment polymorphism (M-AFLP) of the genomic DNAs in the L1 and L2+3 layers of the carnation cultivar, “White Sim”, vegetatively propagated over the past 50 years, were completely different. The bud mutant, “White Mind”, obtained from “White Sim” about 25 years ago, also had very different M-AFLP patterns to the parental “White Sim” line. The cultivar, “Red”, which was separated from “Satisfaction” as a bud mutant two years ago, showed similar patterns to “Satisfaction” in each corresponding layer. However, we were able to detect minor, but significantly different M-AFLP patterns between the cultivars, “Red” and “Satisfaction”. The number of bands that differed between these cultivars was larger in the L2+3 layers than in the L1 layers. The results indicate that the DNA methylation profiles differ between each cell layer derived from distinct cell lineages of vegetatively propagated plants, and that changes in these profiles occur frequently and accumulate in the cells of the L2+3 layers, rather than in the L1 layer.

Transcription regulation of a Kunitz chymotrypsin inhibitor gene which is expressed abundantly in seeds and tuberous roots of winged bean

A winged bean Kunitz-type chymotrypsin inhibitor (WCI) accumulates abundantly in developing embryos and tuberous roots, and also in a small amount in stems. Promoter activity of the 1.0 kbp upstream region of the WCI-3b gene, which encodes a major WCI protein in seeds, was analyzed in transgenic tobacco plants. The 1.0 kbp upstream region directed strong expression of a reporter gene (s-glucuronidase; GUS) in developing seeds of the transgenic tobacco. The GUS activity was also detected in stems, although no significant GUS activity was observed in leaves. Histochemical analysis revealed that the GUS gene was mainly expressed in embryos and in phloem of stems. These results suggested that the transcription regulation plays an important role in the expression of the WCI-3b gene in embryos and in phloem of stems. The cis-elements promoting transcription of the WCI-3b gene in these cells are included within the 1.0 kbp upstream region.