Biomedical subjects

Hong-Gyu Kang

Publications and source records attributed to Hong-Gyu Kang.

12 recordsLinked to original sources

PubMed2006

The rice FON1 gene controls vegetative and reproductive development by regulating shoot apical meristem size.

Most plant organs develop from meristems. Rice FON1, which is an ortholog of Clv1, regulates stem cell proliferation and organ initiation. The point muta-tions, fon1-1 and fon1-2, disrupt meristem balance, resulting in alteration of floral organ numbers and the architecture of primary rachis branches. In this study, we identified two knockout alleles, fon1-3 and fon1-4, generated by T-DNA and Tos17 insertion, respectively. Unlike the previously isolated point mutants, the null mutants have alterations not only of the reproductive organs but also of vegetative tissues, producing fewer tillers and secondary rachis branches. The mutant plants are semi-dwarfs due to delayed leaf emergence, and leaf senescence is delayed. SEM analysis showed that the shoot apical meristems of fon1-3 mutants are enlarged. These results indicate that FON1 controls vegetative as well as reproductive development by regulating meristem size.

Aging↗

PubMed2006

Generation of a flanking sequence-tag database for activation-tagging lines in japonica rice.

We have generated 47,932 T-DNA tag lines in japonica rice using activation-tagging vectors that contain tetramerized 35S enhancer sequences. To facilitate use of those lines, we isolated the genomic sequences flanking the inserted T-DNA via inverse polymerase chain reaction. For most of the lines, we performed four sets of amplifications using two different restriction enzymes toward both directions. In analyzing 41,234 lines, we obtained 27,621 flanking sequence tags (FSTs), among which 12,505 were integrated into genic regions and 15,116 into intergenic regions. Mapping of the FSTs on chromosomes revealed that T-DNA integration frequency was generally proportional to chromosome size. However, T-DNA insertions were non-uniformly distributed on each chromosome: higher at the distal ends and lower in regions close to the centromeres. In addition, several regions showed extreme peaks and valleys of insertion frequency, suggesting hot and cold spots for T-DNA integration. The density of insertion events was somewhat correlated with expressed, rather than predicted, gene density along each chromosome. Analyses of expression patterns near the inserted enhancer showed that at least half the test lines displayed greater expression of the tagged genes. Whereas in most of the increased lines expression patterns after activation were similar to those in the wild type, thereby maintaining the endogenous patterns, the remaining lines showed changes in expression in the activation tagged lines. In this case, ectopic expression was most frequently observed in mature leaves. Currently, the database can be searched with the gene locus number or location on the chromosome at http://www.postech.ac.kr/life/pfg/risd. On request, seeds of the T(1) or T(2) plants will be provided to the scientific community.

Chromosome Mapping↗

PubMed2005

Morphological alterations by ectopic expression of the rice OsMADS4 gene in tobacco plants.

OsMADS4, a rice MADS-box gene, is a member of the GLO/PI family that specifies the identity of petals and stamens in combination with other MADS-box genes. We report here the ectopic expression of OsMADS4 fused to the CaMV 35S promoter in tobacco plants. Transgenic plants carrying the CaMV 35S promoter::OsMADS4 construct generated mutant flowers with a mosaic carpel, in which the tissue around the nectary was elongated and the styles reduced. The fruits were distorted, but viable seeds did develop. These phenotypes mimicked those of transgenic tobacco plants that ectopically express Antirrhinum GLO. However, unlike GLO, OsMADS4 did not cause any homeotic change in the first whorl of the transgenic flowers. These results suggest that the functional role of OsMADS4 in the outer whorls has diverged from that of its dicot counterparts.

Flowers↗

PubMed2005

White-core endosperm floury endosperm-4 in rice is generated by knockout mutations in the C-type pyruvate orthophosphate dikinase gene (OsPPDKB).

We have isolated a floury endosperm-4 (flo4) rice mutant with a floury-white endosperm but a normal outer portion. Scanning electron microscopic analysis revealed that this abnormal endosperm consisted of loosely packed starch granules. The mutant phenotype was generated by T-DNA insertion into the fifth intron of the OsPPDKB gene encoding pyruvate orthophosphate dikinase (PPDK). Plants containing flo4-1 produced no OsPPDKB transcript or the OsPPDKB protein in their developing kernels and leaves. We obtained two additional alleles, flo4-2 and flo4-3, that also showed the same white-core endosperm phenotype. The flo4 kernels weighed about 6% less than wild-type ones. Starch contents in both kernel types were similar, but the total protein content was slightly higher in the mutant kernels. Moreover, lipid contents were significantly increased in the flo4 kernels. Expression analyses demonstrated that the cytosolic mRNA of OsPPDKB was induced in the reproductive organs after pollination, and greatly increased until about 10 days after fertilization. This mRNA was localized mainly in the endosperm, aleurone, and scutellum of the developing kernel. Our results suggest that cytosolic PPDK functions in rice to modulate carbon metabolism during grain filling.

Lipids↗

PubMed2004

Generation of T-DNA tagging lines with a bidirectional gene trap vector and the establishment of an insertion-site database.

We have developed a binary T-DNA vector, pGA2717, that contains the promoter-less beta-glucuronidase (gus) gene adjacent to the right border and the promoter-less green fluorescence protein (gfp) gene next to the left border of the T-DNA. Therefore, inserting T-DNA into a gene can result in the activation of either gus or gfp. A total of 12 169 T-DNA insertional lines of japonica rice were generated using this binary vector. Out of 3140 lines examined, 0.5% of their mature seeds and 2.0% of the 3-day-old etiolated seedlings were GFP-positive. However, GUS assays of the same materials resulted in the identification of 151 (4.8%) GUS-positive lines. Using DNA gel blot and reverse transcription (RT)-PCR analyses, we confirmed that the GFP-positive lines were a true indication of gene trapping. A fusion transcript was also obtained between gfp and the trapped gene. We isolated 990 genomic sequences flanking T-DNA from our analysis of 2099 transgenic plants. Among the insertions, 625 T-DNAs were integrated into genic regions; 361 were located in intergenic regions. These tagging lines will be valuable in trapping and studying various genes for their expression patterns, as well as providing a useful tool for genetic approaches.

Base Sequence↗

PubMed2003

Generation and analysis of end sequence database for T-DNA tagging lines in rice.

We analyzed 6749 lines tagged by the gene trap vector pGA2707. This resulted in the isolation of 3793 genomic sequences flanking the T-DNA. Among the insertions, 1846 T-DNAs were integrated into genic regions, and 1864 were located in intergenic regions. Frequencies were also higher at the beginning and end of the coding regions and upstream near the ATG start codon. The overall GC content at the insertion sites was close to that measured from the entire rice (Oryza sativa) genome. Functional classification of these 1846 tagged genes showed a distribution similar to that observed for all the genes in the rice chromosomes. This indicates that T-DNA insertion is not biased toward a particular class of genes. There were 764, 327, and 346 T-DNA insertions in chromosomes 1, 4 and 10, respectively. Insertions were not evenly distributed; frequencies were higher at the ends of the chromosomes and lower near the centromere. At certain sites, the frequency was higher than in the surrounding regions. This sequence database will be valuable in identifying knockout mutants for elucidating gene function in rice. This resource is available to the scientific community at http://www.postech.ac.kr/life/pfg/risd.

Base Sequence↗

PubMed2003

Transgene structures in T-DNA-inserted rice plants.

T-DNA is commonly used for delivery of foreign genes and as an insertional mutagen. Although ample information exists regarding T-DNA organization in dicotyledonous plants, little is known about the monocot rice. Here, we investigated the structure of T-DNA in a large number of transgenic rice plants. Analysis of the T-DNA borders revealed that more than half of the right ends were at the cleavage site, whereas the left ends were not conserved and were deleted up to 180 bp from the left border (LB) cleavage site. Three types of junctions were found between T-DNA and genomic DNA. In the first, up to seven nucleotide overlaps were present. The frequency of this type was much higher in the LB region than at the right border (RB). In the second type, which was more frequent in RB, the link was direct, without any overlaps or filler DNA. Finally, the third type showed filler DNA between T-DNA and the plant sequences. Out of 171 samples examined, 77 carried the vector backbone sequence, with the majority caused by the failure of T-strand termination at LB. However, a significant portion also resulted from co-integration of T-DNA and the vector backbone to a single locus. Most linkages between T-DNA and the vector backbone were formed between two 3' ends or two 5' ends of the transferred DNAs. The 3' ends were mostly linked through 3-6 bp of the complementing sequence, whereas the 5' ends were linked through either precise junctions or imprecise junctions with filler DNA.

Base Sequence↗

PubMed2003

CvADH1, a member of short-chain alcohol dehydrogenase family, is inducible by gibberellin and sucrose in developing watermelon seeds.

To understand the molecular mechanisms that control seed formation, we selected a seed-preferential gene (CvADH1) from the ESTs of developing watermelon seeds. RNA blot analysis and in situ localization showed that CvADH1 was preferentially expressed in the nucellar tissue. The CvADH1 protein shared about 50% homology with short-chain alcohol dehydrogenase including ABA2 in Arabidopsis thaliana, stem secoisolariciresinol dehydrogenase in Forsythia intermedia, and 3beta-hydroxysterol dehydrogenase in Digitalis lanata. We investigated gene-expression levels in seeds from both normally pollinated fruits and those made parthenocarpic via N-(2-chloro-4-pyridyl)-N'-phenylurea treatment, the latter of which lack zygotic tissues. Whereas the transcripts of CvADH1 rapidly started to accumulate from about the pre-heart stage in normal seeds, they were not detectable in the parthenocarpic seeds. Treating the parthenogenic fruit with GA(3) strongly induced gene expression, up to the level accumulated in pollinated seeds. These results suggest that the CvADH1 gene is induced in maternal tissues by signals made in the zygotic tissues, and that gibberellin might be one of those signals. We also observed that CvADH1 expression was induced by sucrose in the parthenocarpic seeds. Therefore, we propose that the CvADH1 gene is inducible by gibberellin, and that sucrose plays an important role in the maternal tissues of watermelon during early seed development.

Alcohol Dehydrogenase↗

PubMed2003

Systematic reverse genetic screening of T-DNA tagged genes in rice for functional genomic analyses: MADS-box genes as a test case.

We have generated 47 DNA pools and 235 subpools from 21,049 T-DNA insertion lines of rice. DNA pools of 500-1,000 lines were adequate for screening a T-DNA insertion within a 2-kb region. To examine the efficacy of the DNA pools, we selected MADS-box genes, which play an important role in controlling various aspects of plant development. A total of 34 MIKC-type MADS-box genes have now been identified from rice sequence databases. Our PCR screening for T-DNA insertions within 12 MADS-box genes resulted in the identification of five insertions in four different genes. These DNA pools will be valuable when isolating T-DNA insertional mutants in various rice genes. The DNA pool screening service and the mutant seeds are available upon request to genean@postech.ac.kr.

DNA, Bacterial↗

PubMed2002

Molecular characterization of a GA-inducible gene, Cvsus1, in developing watermelon seeds.

To understand the molecular mechanisms that control seed development, we isolated a seed-preferential gene from ESTs of developing watermelon seeds. The gene Cvsus1 encodes a protein that is 86% identical to the Vicia faba sucrose synthase expressed in developing seeds. RNA blot analysis showed that Cvsus1 was preferentially expressed in watermelon seeds. We also investigated gene expression levels both in pollinated seeds and in parthenocarpic seeds, which lack zygotic tissues. Whereas the transcript level of Cvsus1 was rapidly increased during normal seed development, the expression was not significantly increased in the parthenocarpic seeds. However, treating the parthenocarpic fruits with GA3 strongly induced Cvsus1 expression, up to the level accumulated in pollinated seeds. These results suggest that Cvsus1 is induced in maternal tissues via signals from the zygotic tissues, and that GA may be one of those signals.

Blotting, Southern↗

PubMed2002

Cloning of gibberellin 3 beta-hydroxylase cDNA and analysis of endogenous gibberellins in the developing seeds in watermelon.

We have isolated Cv3h, a cDNA clone from the developing seeds of watermelon, and have demonstrated significant amino acid homology with gibberellin (GA) 3 beta-hydroxylases. This cDNA clone was expressed in Escherichia coli as a fusion protein that oxidized GA(9) and GA(12) to GA(4) and GA(14), respectively. The Cv3h protein had the highest similarity with pumpkin GA 2 beta,3 beta-hydroxylase, but did not possess 2 beta-hydroxylation function. RNA blot analysis showed that the gene was expressed primarily in the inner parts of developing seeds, up to 10 d after pollination (DAP). In the parthenocarpic fruits induced by treatment with 1-(2-chloro-4-pyridyl)-3-phenylurea (CPPU), the embryo and endosperm of the seeds were undeveloped, whereas the integumental tissues, of maternal origin, showed nearly normal development. Cv3h mRNA was undetectable in the seeds of CPPU-treated fruits, indicating that the GA 3 beta-hydroxylase gene was expressed in zygotic cells. In our analysis of endogenous GAs from developing seeds, GA(9) and GA(4) were detected at high levels but those of GA(20) and GA(1) were very low. This demonstrates that GA biosynthesis in seeds prefers a non-13-hydroxylation pathway over an early 13-hydroxylation pathway. We also analyzed endogenous GAs from seeds of the parthenocarpic fruits. The level of bioactive GA(4 )was much lower there than in normal seeds, indicating that bioactive GAs, unconnected with Cv3h, exist in integumental tissues during early seed development.

Amino Acid Sequence↗

PubMed2002

T-DNA insertional mutagenesis for activation tagging in rice.

We have developed a new T-DNA vector, pGA2715, which can be used for promoter trapping and activation tagging of rice (Oryza sativa) genes. The binary vector contains the promoterless beta-glucuronidase (GUS) reporter gene next to the right border. In addition, the multimerized transcriptional enhancers from the cauliflower mosaic virus 35S promoter are located next to the left border. A total of 13,450 T-DNA insertional lines have been generated using pGA2715. Histochemical GUS assays have revealed that the GUS-staining frequency from those lines is about twice as high as that from lines transformed with the binary vector pGA2707, which lacks the enhancer element. This result suggests that the enhancer sequence present in the T-DNA improves the GUS-tagging efficiency. Reverse transcriptase-PCR analysis of a subset of randomly selected pGA2715 lines shows that expression of the genes immediately adjacent to the inserted enhancer is increased significantly. Therefore, the large population of T-DNA-tagged lines transformed with pGA2715 could be used to screen for promoter activity using the gus reporter, as well as for creating gain-of-function mutants.

Caulimovirus↗