• Journal of Korean Society of Forest Science
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• v.86 no.4
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• pp.407-414
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• 1997

The resistance of a non-transgenic poplar clone, 'Ogy' and three transgenic poplar lines to the cottonwood leaf beetle, Chrysomela scripta F., was evaluated by in vitro feeding. The lines were transformed with neomycin phosphotransferase II(NPT II) as a selectable marker, proteinase inhibitor II(pin2) as a resistance gene, and CaMV 35S as a promoter. An efficient method of sterilizing the beetle eggs and introducing them into plant tissue cultures was developed. The resistance of the transgenic lines was investigated in terms of effects tin leaf area consumed, insect weight, insect developmental stages, and plantlet root dry weight after feeding. Also, leaf area consumed was examined by leaf age as measured through leaf plastochron index(LPI). The leaf area consumed and insect weight were highly significant between transformants and control, and insect development in vitro was significant among the transgenic lines. Larval infestation was the most severe around LPI 4 to 5 which were young leaves. The system provided a quick, highly controlled method to screen developing transgenic plantlets directly.

• Applied Biological Chemistry
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• v.39 no.2
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• pp.104-111
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• 1996

We have isolated several proteinase inhibitor II genes pin2 from a Russet Burbank potato DNA library. One of these, pin2T was subcloned and a 1.8 kb Xbal/Nsil insert was sequenced. This fragment contained the complete Inhibitor II gene including 965 Up of flanking DNA upstream from the gene and 200 bp of flanking DNA downstream from the gene. The open reading frame encodes a protein that is similar to other reported proteinase Inhibitor II proteins. The DNA sequence of the 5' flanking region of pin2T from -714 to +1 is highly homologous (91% identity) with that of the previously isolated wound-inducible pin2K. There are, however, four small deletions in the pin2T promoter which are located at -221 to -200, -263 to -254, -523 to -426 and -759 to -708 relative to the transcription start site of the wound-inducible pin2K. Three of these deletions map to a portion of the promoter that controls the wound-inducibility of the proteinase inhibitor genes. Chimeric genes containing the promoter of the pin2T gene linked with the both CAT and GUS were constructed and transfered into tobacco plants. Analysis of these plants indicated that pin2T is not a wound-inducible gene but is expressed at low levels. Thus, wound-inducibility is lost with the concomitant natural deletion of three small regions of the promoter. Comparision of the sequences deleted in pin2T relative to the pin2K with Genebank sequences indicates that the deleted sequences contain a motif (consensus 5'-AGTAAA-3') that is found in many other wound-inducible genes but not easily found in the published promoter sequences of other plant genes. Nuclear proteins from unwounded and wounded potato leaves were bound to the proximal promoter region, downstream of the 5'-AGTAAA-3', of pin2T. The comparison of the pin2T gone with the pin2K gene indicates that the natural internal promoter deletions are likely responsible for loss of the wound-inducible phenotype in the pin2T gene.

• Journal of Applied Biological Chemistry
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• v.43 no.1
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• pp.12-17
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• 2000

To understand the signal transduction pathway that leads to the activation of the wound-inducible proteinase inhibitor II (pin2) promoter. $F_2$ progenies of wound (-) mutant crossed with wild-type Arabidopsis plants were biochemically and genetically characterized. Wound (-) mutant was derived from transgenic Arabidopsis plants containing bacterial cytosine deaminase gene under the control of pin2 promoter. The cytosine deaminase assays indicated that wound (-) mutant is a dominant inhibitor of wound-inducibility as only 3 of the $20F_2$ progenies showed cytosine deaminase (CDase) activity, To construct a structural map of the wound (-) mutant chromosomal regions, cleaved, amplified polymorphic sequences (CAPS) markers that cover all Chromosomes were used. Chromosomal regions covered by three different CAPS markers could be candidates for further fine mapping of the location of the wound (-) mutation. g4026, RGA1 and ASA1 located at 84.9 on recombinant inbred (RI) map of chromosome I, at 1.75 on RI map of chromosome II, and 18.35 on RI map of chromosome V, respectively.

• Applied Biological Chemistry
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• v.40 no.4
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• pp.271-276
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• 1997

We have prepared several chimeric constructs containing the bacteriophage T7 RNA polymerase gene under control of the wound-inducible potato proteinase inhibitor II (pin2) promoter and have transformed Nicotiana tabacum plants with these constructs. Southern blot analyses indicate that either one or two copies of the gene constructs are present in the transgenic plants. Northern blot analyses indicate that mRNA encoding T7 RNA polymerase is expressed in a wound-inducible manner. We purified T7 RNA polymerase and prepared antiserum. This antiserum was used for Western blot analyses to demonstrate that a protein which is cross reactive with T7 RNA polymerase is produced. The molecular mass of this protein is 80 kDa, a size which is consistant with the nicked form of the polymerase as is often seen when expressed in E. coli. RNA polymerase assays were used to indicate that the nicked form of T7 RNA polymerase is active and capable of incorporating labeled nucleotides into transcripts in vitro. Analysis of transgenic plants did indeed show that wound-inducible activation of the T7 RNA polymerase permits the establishment of a genetic system to overexpress genes in plants using T7 RNA polymerase(Received March 20, 1997; accepted May 2, 1997)

• Journal of Korean Society of Forest Science
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• v.84 no.1
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• pp.77-86
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• 1995

To effectively modify tree function with genetic engineering, transgenes must be expressed at the proper level in the appropriate tissues at suitable developmental stages. Toward understanding the spatial and temporal expression of transgenes in woody plants, transgene expression was evaluated in three greenhouse-grown, transgenic lines of Populus alba ${\times}$ P. grandidentata hybrid clone 'Hansen'. All transgenic poplar lines possess constructs containing the bacterial nopaline synthase(nos) promoter linked to a neomycin phosphotransferase II(NPT II) selectable marker gene. In addition, each transgenic poplar line contains one of the following gene constructs : 1) a wound-inducible potato proteinase inhibitor II (pin2) promoter linked to a chloramphenicol acetyltransferase(CAT) reporter gene. 2) a nos promoter linked to a PIN2 structural gene : or 3) a Cauliflower Mosaic Virus 35s promoter linked to a PIN2 structural gene. Polymerase chain reaction(PCR) was used to verify the presence of foreign genes in the poplar genome. Enzyme-linked immunosorbent assays(ELISAs) were used to evaluate organ specific expression of the nos-NPT II construct. NPT II expression was detected in leaves, petioles, stems, and roots of transgenic poplar, thereby indicating that the nos promoter is potentially effective for general constitutive expression of transgenes. NPT expression varied among transgenic poplar lines and among organs for one transgenic line, Tr15. With Tr15, NPT II levels were highest in older leaves and petioles. These results indicate that screening of several transgenic lines may be required to identify lines with optimal transgene expression.

• Journal of Plant Biotechnology
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• v.47 no.2
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• pp.157-163
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• 2020

Calmodulin (CaM) mediates cellular Ca²⁺ signals in the defense responses of plants. We previously reported that GmCaM-4 and 5 are involved in salicylic acid-independent activation of disease resistance responses in soybean (Glycine max). Here, we generated a GmCaM-4 cDNA construct under the control of the cauliflower mosaic virus (CaMV) 35S promoter and transformed this construct into potato (Solanum tuberosum L.). The constitutive over-expression of GmCaM-4 in potato induced high-level expression of pathogenesis-related (PR) genes, such as PR-2, PR-3, PR-5, phenylalanine ammonia-lyase (PAL), and proteinase inhibitorII (pinII). In addition, the transgenic potato plants exhibited enhanced resistance against a bacterial pathogen, Erwinia carotovora ssp. Carotovora (ECC), that causes soft rot disease and showed spontaneous lesion phenotypes on their leaves. These results strongly suggest that a CaM protein in soybean, GmCaM-4, plays an important role in the response of potato plants to pathogen defense signaling.