Young-Doo Park

Homology-dependent gene silencing in transgenic plants: epistatic silencing loci contain multiple copies of methylated transgenes

Previous work has shown that two homologous, unlinked transgene loci can interact in plant nuclei, leading to non-reciprocal trans-inactivation and methylation of genes at one locus. Here, we report the structure and methylation of different transgene loci that contain the same construct but are variably able to inactivate and methylate a partially homologous, unlinked target locus. Silencing loci comprised multiple, methylated copies of the transgene construct, whereas a non-silencing locus contained a single, unmethylated copy. The correspondence between strength of silencing activity and copy number/degree of methylation was further demonstrated by producing novel alleles of a strong silencing locus: reducing the transgene copy number and methylation within this silencing locus decreased its ability to inactivate the target locus. The strong silencing locus, which was located close to a telomere, trans-inactivated various structural variants of the original target construct, regardless of their location in the genome. This suggests that the silencing locus can scan the entire genome for homologous regions, a process possibly aided by its telomeric location. Our data support the idea that epistatic trans-inactivation of unlinked, homologous transgenes in plants results from a pre-existing epigenetic difference between transgene loci, which is subsequently equalized by “epigene conversion” involving DNA-DNA pairing.

Susceptibility of transgene loci to homology-dependent gene silencing.

Previous work has shown that two unlinked, partially homologous transgene loci can interact in plant nuclei, leading to reversible methylation and inactivation of one transgene locus in the presence of the second. To study whether the chromosomal location of a transgene influences its susceptibility to trans-inactivation, we retransformed four transgenic lines, which contained the same construct (H) integrated in different chromosomal locations, with a second, partially homologous construct (K). At least 50 double transformants (DTs) were regenerated from each single transformant (ST) and screened for inactivation of markers [chloramphenicol acetyltransferase (CAT); hygromycin resistance (HYGR)] at the resident H locus. For two STs, H locus markers were inactivated in less than 1% of the DTs, suggesting that, at these integration sites, H was relatively resistant to trans-inactivation. In contrast, the other two STs appeared to be more sensitive to trans-inactivation: 4–10% of the DTs were CAT− and/or HygS. Inactivation of H locus markers could be attributed to two distinct phenomena:1.Regeneration from cells containing different epigenetic states of H, in which either both, one or none of the H alleles was active. This instability in the expression of the H locus, which was independent of K, was more pronounced in the homozygous state, and was associated with cellular mosaicism of expression and methylation.2.The presence of an unlinked K locus could weaken the HygR phenotype by transcriptional inactivation and increased methylation of the hph gene at the H locus. These results indicated that a susceptible transgene locus is inherently unstable and partially methylated, and that these characteristics are exacerbated when the locus is homozygous for the transgene and/or when an unlinked homologous transgene is present.

Effects of Korean red ginseng and its mixed prescription on the high molecular weight dextran-induced blood stasis in rats and human platelet aggregation

This study was undertaken to evaluate the antithrombotic effects of Korean Red Ginseng (KRG) and its new prescription (KRGP) consisting of five herbs such as Korean red ginseng, Ganoderma, Cinnamomi Cortex, Glycyrrhizae Radix and Laminaria. In rats with blood stasis induced by high molecular weight dextran, KRG and KRGP significantly restored not only the number of platelets and fibrinogen, but also suppressed the fibrin degradation products (FDP) to normal range. In platelet aggregation assay with human platelet rich plasma (PRP), KRG and KRGP significantly inhibited thrombin and collagen-induced platelet aggregation. The IC(50) values of KRG and KRGP were >2 and 0.23+/-0.01 mg/ml for thrombin, 0.32+/-0.01 and 0.17+/-0.02 mg/ml for collagen and 0.72+/-0.25 and >2 mg/ml for ADP, respectively. In coagulation assay, KRG and KRGP significantly prolonged activated partial prothrombin time (APPT) and prothrombin time (PT) as compared with control data. KRGP was found to be more effective than KRG alone on antithrombotic activity. These results suggest that KRGP may exert its antithrombotic activity due to inhibition of platelet aggregation and coagulation activity more than KRG.

Agrobacterium-Mediated Transformation System for Large-Scale Producion of Transgenic Chinese Cabbage (Brassica rapa L. ssp. pekinensis) Plants for Insertional Mutagenesis

In order to better utilize insertional mutagenesis and functional genomics in Chinese cabbage, we have developed an improved transformation system that more efficiently produces a large number of transgenic plants. Hypocotyl explants were inoculated withAgrobacterium tumefaciens LBA4404. This strain harbors tagging vector pRCV2, which contains a hygromycin-resistance gene, an ampicillin resistance gene, and a bacterial replication origin within the T-DNA. Transformation efficiency was highest when the explants were first co-cultivated for 3 d in a medium supplemented with 5 mg L-1 acetosyringone, then transferred to a 0.8% agar selection medium containing 10 mg L-1 hygro-mycin. In addition, maintaining a low pH in the co-cultivation medium was critical to enhancing transformation frequency. A total of 3369 transgenic plants were obtained, with efficiencies ranging from 2.89% to 5.00%. Southern blot analysis and T, progeny tests from 120 transgenic plants confirmed that the transgenes were stably inherited to the next generation. We also conducted plasmid rescue and inverse PCR with some transformants, based on their phenotype, to demonstrate the applicability of T-DNA tagging in Chinese cabbage. The tagged sequences were then analyzed.

Inheritance and expression of a transgene insert in an aneuploid tobacco line

A T-DNA locus comprising nptII, uidA and nos genes — all under the control of the nos promoter (this locus was designated K because it encodes resistance to Kanamycin) - was found to be inherited erratically in a transgenic tobacco line. This anomalous behavior was partially explained following a karyotype analysis of plants representing several generations: these plants were aneuploids, presumably for the K-containing chromosome. During four generations of sexual propagation, transgenic plants that were either trisomic or tetrasomic for the K-containing chromosome (i.e. 2n=49 or 2n=50, respectively) were obtained. The trisomic plants (2n=48+1) were virtually indistinguishable phenotypically from normal euploids (2n=4x=48), whereas the tetrasomic plants (2n=48+2) were smaller, had somewhat misshapen leaves and exhibited reduced fertility. Although the amount of NPTH protein in different trisomic (K--, KK-, KKK) and tetrasomic (KK--, KKK-) plants was generally consistent with a K dosage effect, the genetic behavior of each trisomic — with respect to segregation of KanR and marker gene activity in progeny — was unique and not completely explicable by invoking aneuploidy. Specifically, unexpected gains or losses of K could occur, suggesting the formation of double reductional gametes and/or frequent gene conversion at this locus. The susceptibility of K locus marker genes to trans-inactivation in the trisomic and tetrasomic lines was tested by crossing in partially homologous silencing loci. In all transgenotypes tested, the three K marker genes were sensitive to trans-silencing, which was accompanied by methylation in all copies of the nos promoter. In addition to this directed inactivation/methylation, the K locus could also undergo infrequent, spontaneous partial methylation, which produced stable epialleles. In most plants, however, the multiple copies of the nos promoter at this locus remained unmethylated and active through four generations in all transgenotypes examined. The significance of these results for irregular inheritance patterns, aneuploid syndromes and homology-dependent gene silencing is discussed.

Plant regeneration from leaf tissues of four North Dakota genotypes of potato (Solanum tuberosum L.)

An efficient plant regeneration system was developed fromin vitro leaf tissues of four North Dakota potato genotypes. The best medium for genotype ND860-2 was Murashige and Skoog medium with 20.0 μM IAA and 11.4 μM zeatin riboside. Two to three weeks of initial dark treatments had a significant effect in reducing the time for shoot regeneration and increasing the number of regenerated shoots. Four antibiotics commonly used inAgrobacterium- mediated transformation were tested for their effects on shoot regeneration. Shoot induction was completely inhibited by kanamycin at 15 mg/L and hygromycin at 4 mg/L or higher, but not by carbenicillin (except at 1000 mg/L) and cefotaxime. Hygromycin significantly stimulated shoot induction at 1 mg/L.CompendioSe desarrolló un sistema eficiente de regeneratión vegetal utilizando tejidos in vitro de hojas de cuatro genotipo de papa de North Dakota. El mejor medio para el genotipo ND860-2 fue el de Murashige y Skoog con 20.0 μM IAA y 11.4 μM de zeatin riboside. Tratamientos iniciales de dos a tres semanas en la oscuridad tuvieron un efecto signifïcativo en reducir el tiempo para la regeneratión del brote y en el incremento del número de brotes regeneradores. Se probaron cuatro antibióticos comunmente utilizados en la transformatión por medio del Agrobacterium, para sus efectos sobre la regeneratión de brotes. La induction de brotes fue completamente inhibida por kanamycin a 15 mg/L e hygromycin a 4 o más mg/L, pero no por carbenicillin (con la exceptión a 1,000 mg/L) y cefotaxime. A1 mg/L, hygromycin estimuló signifïcativamente la inductión de brotes.

13-Hydroxy-9Z,11E,15E-octadecatrienoic acid from the leaves of Cucurbita moschata.

A new unsaturated hydroxy fatty acid was isolated from the leaves ofCucurbita moschata through repeated silica gel column chromatography and chemical methods. The structure of the new fatty acid was determined as 13-hydroxy-9, 11, 15-octadecatrienoic acid on the basis of several spectral data including 2D-NMR. The stererostructures of double bonds were determined to be 9Z, 11E and 15E by coupling patterns of related proton signals in the1H-NMR and NOESY experiments.

A non-antibiotic selection system uses the phosphomannose-isomerase (PMI) gene forAgrobacterium-mediated transformation of Chinese cabbage

To establish a non-antibiotic selection system that utilizes the phosphomannose-isomerase (PMI) gene for Chinese cabbage transformation, we first determined the optimum mannose concentration for selecting transformed cells. Hypocotyl and cotyledon expiants that were grown on media containing more than 5 g L-1 mannose did not induce green calli but, rather became chlorotic and withered before dying. In contrast, media containing 20 g L-1 sucrose plus 5 g L-1 mannose proved suitable for selection. We then used this particular level of mannose to transform hypocotyl tissues. Within 6 weeks, shoots were regenerated from some of the calli; subsequently, these plants were transplanted to pots and grown in the greenhouse. A 514-bp PCR fragment was obtained from most transformants but not from the non-transformed plants. Southern blot analysis also revealed the expectedPMI gene in those PCR-confirmed transgenic plants. RT-PCR of total RNA was performed to confirmPMI expression. We have now demonstrated that this gene does not inhibit the growth of transgenic plants, and that this selection system can be applied to Chinese cabbage transformation.

Development of anAgrobacterium-mediated transformation system for regenerating garland chrysanthemum (Chrysanthemum coronarium L.)

Although efficient shoot regeneration and selection are essential for genetic transformation mediated byAgrobacterium, success has been limited with the garland chrysanthemum (Chrysanthemum coronarium L.). In this study, we developed a useful protocol for shoot regeneration with leaf disk explants. The optimal concentrations of NAA and BA were 0.2 mg L−1 and 0.5 mg L−1, respectively. To optimize the selection system for regenerating plants from genetically transformed tissues, we tested the effects of four antibiotics (kanamycin, hygromycin, carbenicillin, and cefotaxime). Among them, 5 mg L-1 hygromycin proved adequate as a selectable marker, whereas 500 mg L-1 carbenicillin was effective in eliminating excessiveAgrobacterium after co-cultivation. Transgenic plants were obtained by first co-culturing garland chrysanthemum leaf disks withA. tumefaciens strain EHA105, which harbors plasmid pRCVII containing the hygromycin resistance (hpt) and β-glucuronidase (GUS) genes. After the transgenic plants were confirmed via Southern analysis, they were rooted in soil and appeared phenotypically normal. Our report is the first to describe the optimum conditions for producing transgenic plants of this species.

Studies on the effects of a flanking repetitive sequence on the expression of single-copy transgenes in Nicotiana sylvestris and in N. sylvestris-N. tomentosiformis hybrids

To test the influence of a Nicotiana tomentosiformis repetitive sequence (R8.3) on transgene expression in N.xa0sylvestris and in N.xa0sylvestris-N.xa0tomentosiformis hybrids, the R8.3 sequence was placed upstream of a nopaline synthase promoter (NOSpro)-NPTII reporter gene in a T-DNA construct. A number of transgenic N.xa0sylvestris lines were produced and in most, the NPTII gene was expressed. In one line, however, the NPTII gene became silenced and methylated in the NOSpro region. The silenced locus was able to trans-inactivate and induce methylation of two stably expressed transgene loci comprising a similar construct. Nucleotide sequence analyses of the three transgene loci revealed that they each contained a single incomplete copy of the T-DNA, which had sustained deletions of varying sizes in the R8.3 region. Paradoxically, the R8.3 DNA upstream of the two active, unmethylated NOSpro-NPTII genes was highly methylated, whereas the R8.3 DNA upstream of the silenced, methylated NOSpro-NPTII gene was less methylated. The methylated portions of the R8.3 sequence corresponded to retroelement remnants. An active NOSpro-NPTII gene downstream of a nearly intact R8.3 sequence did not become methylated in N.xa0sylvestris-N.xa0tomentosiformis hybrids. Thus, methylation in the R8.3 sequence did not spread into adjoining transgene promoters and the effect of the R8.3 dispersed repeat family on transgene expression was negligible. The silencing phenomena observed with the three single-copy transgene loci are discussed in the context of other possible triggers of silencing.