P-1 G. Suzuki1, T. Ohta1, M. Yamamoto2,
M. Morell3, S. Rahman3, and Y Mukai.1 (1Osaka
Kyoiku Univ, 2Kansai Women's Col, 3CSIRO)
Genomic organization of the SBEI multigene family in the D genome
donor of wheat. II.
A wheat SBEI (starch branching enzyme I) multigene family is related to synthesis of starch. We isolated 120-kb and 100-kb clones containing SBEI genes from a large-inserts genomic library of Aegilops squarrosa, D genome donor of wheat. DNA gel blot of the clones with several different SBEI probes indicated that three SBEI genes were clustered within 100 kb. Sequence analysis of subclones and lambda clones demonstrated that wSBEI-D2 was located 3-kb downstream of wSBEL-D3, and 3-kb upstream of wSBEI-D4 gene; of these only wSBEI-D4 encodes the SBEI purified from the endosperm. The possible existence of other SBEI-related sequences near the cluster was also suggested by genomic DNA gel blot analysis. We tried to visualize tandem organization of the SBEI multigene family by FISH on circular DNA fibers of the large insert clones.
P-2 T. Shimizu1, A. Meguro1, S. Takumi2,
Y. Ogihara3, and K.Murai1 (1Dep Biosci,
Fukui Pref Univ, 2Fac Agr, Kobe Univ, 3Kihara Inst Biol
Res, Yokohama City Univ)
Isolation of homoeologous genes of wheat AGAMOUS homologue WAG,
which are located on 1A, 1B and 1D chromosomes
Homeotic transformation of stamens into pistil-like structures (pistillody) has been observed in alloplasmic lines of common wheat with the cytoplasm of a wild relative species, Aegilops crassa. To investigate the molecular mechanism of the induction of pistillody, we isolated a MADS box gene WAG (wheat AGAMOUS) from a cDNA library of young spikes, which shows high homology to AGAMOUS. AGAMOUS is class C MADS box gene specifying the identity of stamens and carpels in Arabidopsis. Northern blot analysis revealed that there are two WAG transcripts in the wheat floral organs, one of which is expressed specifically in pistil as well as in the pistillate stamens of the alloplasmic lines. Because Southern blot analysis showed that WAG presented three homoeologous genes, which located on homoeologous group 1 chromosomes (1A, 1B, 1D), we hypotheses that the pistd-specific WAG transcript is derived from one of three WAG homoeologous genes. To verify this hypothesis, we are now cloning and characterizing genomic clones of WAG.
P-3 E. Hama1, S. Takumi2, Y. Ogihara3 and
K. Murai1(1Dep Biosci, Fukui Pref Univ, 2Fac
Agr, Kobe Univ, 3Kihara Inst Biol Res, Yokohama City Univ)
Class B MADS box genes are associated with the induction of pistillody in
alloplasmic wheats
It is well known that class B MADS box genes are involved in specifying the identity of petals and stamens in dicot species. To investigate molecular mechanism of pistillody, homeotic transformation of stamens into pistil-like structures, in alloplasmic wheats with Aegilops crassa cytoplasm, we isolated three cDNA clones of PISTILLATA type class B MADS box genes, WPI-2 (wheat PISTILLATA #2), WPI- 6 and WPI-7. Among these clones, WPI-2 was highly homologous to OsMADS4, rice ortholog of PISTILLATA, and preferentially expressed in young spikes, suggesting that WPI-2 is wheat ortholog of PISTILLATA. In situ hybridization analysis revealed that WPI-2 is expressed in stamen primordia of normal lines, but not in pistillate stamens of the alloplasmic line. These results indicate that pistillody in the alloplasmic wheats is caused by a change of WPI-2 gene expression pattern.
P-4 F. Kobayashi1, S. Takumi1, R. Ohno1,
T. Nakamura2and C. Nakamura1 (1Fac
Agr, Kobe Univ, 2Tohoku Nat Agr Cent)
Low-temperature inducibility of the RAB gene family in wheat
seedlings
To investigate contribution of the wheat RAB (responsive to ABA) gene family to cold acclimation and freezing tolerance, expression patterns of the four RAB genes, Wrab15, Wrab17, Wrab18 and Wrab19, were monitored under cold- and ABA-treated conditions in seedlings of two wheat cultivars Chinese Spring (CS) and Mironoyskaya 808 (M808). Wrab19 and Wrab17 were isolated from cold-treated M808 cDNA library, and Wrab15 and Wrab18 were from an EST library of wheat developing embryos. Wrab18 is likely a homoeologue of Wrab19, based on their nucleotide sequence similarity. mRNAs of Wrab18 and Wrab19 accumulated more in the roots than in the leaves under the low-temperature condition. After ABA-treatment the transcript appeared within 5 hours in M808 leaves but no transcript appeared in CS leaves. Wrab17 mRNA appeared in the above-ground tissues within a day of cold treatment in both cultivars and reached a maximum level within 3 days in M808, while a maximum level reached at the 7th day in CS. The Wrab17 mRNA accumulation was maintained throughout at least two months of cold treatment. In M808 seedlings treated with ABA, the Wrab17 transcript accumulated to reach a maximum level within 1 hour. As to Wrab15, a barley HVA22 orthologue, the transcript accumulated more in the roots than in the leaves under the cold condition. The Wrab15 expression was not induced by ABA-treatment in the leaves of both cultivars. A genomic clone of Wrab17 was isolated.