• 생명과학회지
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• 제15권2호
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• pp.176-181
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• 2005

Calmodulin 유전자의 발현 조절을 연구하기 위해, 벼 calmodulin promoter (RCaM-2)를 분리하여 GUS (report 유전자)에 융합하였다. GUS 활성은 정단조직, 근단 및 관다발 영역과 같은 성장조직에서 높게 발현되었다. 줄기와 페티올의 transverse 절단부위 GUS 활성은 관다발계의 안쪽에 제한되었으며 관다발계의 외부에 위치한 피층과 표피에서는 강하게 발현된 식물에서도 GUS 활성이 나타나지 않았다. GUS 활성은 어린 조직에서 특이적으로 발현되었으며 상처에 의해서 신속하게 증가하였다. RCaM-2 promoter는 세포분열이 왕성한 어린조직이나 생장점에서 강하게 발현되며 mechanical 신호에 의해서 현저히 유도되었다. 이러한 결과는 RCaM-2 유전자의 5'-flanking 영역이 상처에 의해서 유도되는 발현을 조절하는 것으로 추정된다.

• Journal of Plant Biotechnology
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• 제4권4호
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• pp.151-154
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• 2002

Conditions for lily pollen growth in vitro and transformation were optimized. Active pollen tube development was achieved effectively in a medium containing 7% sucrose with pH adjusted to 5.7 at the temperature of 27$^{\circ}C$ for about 16-24 hours. Pollen growth was little impaired by the presence of kanamycin at concentration up to 100 mg/L. Pollen rains near the beginning of germination stage were more reliable for Agrobacterium-mediated GUS DNA transformation via vacuum infiltration lasted for 20-40 minutes. GUS DNA integration and its expression in fully developed pollen tubes could be confirmed by Southern blot hybridization, RT-PCR and histochemical staining.

• Journal of Plant Biology
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• 제37권3호
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• pp.371-380
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• 1994

To determine the patterns and the levels of expression of the cauliflower mosaic virus (CaMV 35S) promoter and the rice actin 1 (Act1) promoter in rice, transgenic rice plants containing CaMV 35S-$\beta$-glucuronidase (GUS) and Act1-GUS constructs were generated and examined by fluorometric and histochemical analyses. The fluorometric analysis of stably transformed calluses showed that the activity of the rice Act1 promoter was stronger than that of the CaMV 35S promoter in rice cells. In a histochemcial study of the transgenic rices, it was shown that the GUS activity directed by the CaMV 35S promoter was localized mainly in parenchymal cells of vascular tissues of leaves and roots and mesophyll cells of leaves. These results are similar to those of potato, a dicot plant. In contrast, rice plant transformed with Act1-GUS fusion construct revealed strong GUS activity in parenchymal cells of vascular tissue, mesophyll cells, epidermal cells, bulliform cells, guard subsidiary cells of leaves and most cells of the root, suggesting that the rice Act1 promoter is more constitutive than the CaMV 35S promoter. It was also confirmed that in both types of transgenic rice little or no staining was localized in metaxylen tracheary elements of vascular tissue from leaves or roots. These results indicate that the rice Act1 promoter can be utilized more successfully for expression of a variety of foreign gene in rice than the CaMV 35S promoter.

• 현장농수산연구지
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• 제25권4호
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• pp.138-147
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• 2024

본 연구는 국내 밀 43 품종에 대한 A. tumefaciens 형질 전환 효율을 검정하기 위해 GUS staining 분석을 수행한 것으로서 대부분의 밀 형질전환 연구가 특정 품종에 국한되어 있기 때문에 국내 장려 밀 품종에 대한 형질전환 효율에 영향을 미치는 조건에 대한 검정이 필요하다. 국내 밀 품종 중 32개에서 1개 이상의 조직에 염색된 신호가 관찰되었으며 4개 품종에서는 염색된 신호가 관찰되지 않았고 6개 품종에서 과도한 A. tumefaciens 성장이 관찰되었고, 7개 품종은 미성숙배의 크기 등의 이유로 GUS staining 분석이 불가능하여 추가 분석에서 제외하였다. 국내 밀 품종별 A. tumefaciens 감염 효율 비교 결과, 백립계 8개 품종 및 적립계 28개 품종에서는 평균적으로 유의한 차이가 없었다. 특히 조농밀, 조품밀, 새올밀, 조중밀, 새금강밀 등 적립계 품종에서 90% 이상의 높은 감염 효율이 관찰되었고, 전체 품종 중 22개는 50% 이상의 효율을 나타내었다. 본 연구를 통하여 조농밀 및 새올밀은 미성숙배에서 전체 조직에서 강한 GUS 발현으로 형질전환에 적합한 품종으로 나타났으며 금강 품종은 백립계에서 상대적으로 높은 발현을 보여 A. tumefaciens을 이용한 형질전환에 적합한 것으로 확인되었다. 추가적인 연구를 통해 품종의 조직배양 효율을 검정하고, 안정적인 GUS 발현 효율을 조사하는 것이 필요하며, 이를 통해 밀 형질전환에 이용 가능한 품종을 더욱 정밀하게 선발할 수 있을 것으로 판단된다. 해당 연구 결과는 국내 밀품종의 형질전환 효율을 높이기 위한 기초 자료로 활용 가능할 것이다.

• International Journal of Oral Biology
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• 제33권3호
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• pp.83-89
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• 2008

Glutamate-induced cobalt uptake reveals non-NMDA glutamate receptors (GluRs) in rat taste bud cells. Previous studies suggest that glutamate-induced cobalt uptake in taste cells occurs mainly via kainate type GluRs. Cobaltstained cells were immunoreactive against GluR6 and KA1 subunits of GluRs. However, the functions of those type of receptors are not known yet. It is important question which types of taste cells are cobalt-stained when stimulated by glutamate and whether they express these kinds of GluRs. Circumvallate and foliate papilla of Sprague-Dawley rats (45-60 days old) were used. A cobalt-staining technique combined with immunohistochemistry against specific markers for taste bud cell types, such as blood group H antigen (BGH), $\alpha$-gustducin (Gus), or neural cell adhesion molecule (NCAM) was employed. We also performed double labeling of GluR6 or KA1 subunits of GluR with each specific marker for taste bud cell types. Lots of cobaltstained taste bud cells expressed Gus-like immunoreactivity, and subsets of the cobalt stained cells appeared NCAM- or BGH-like immunoreactivity. Stimulation with 1 mM glutamate significantly increased the number of cobaltstained cells in Gus-like immunoreactive cells, but not in NCAM- or BGH-like immunoreactive cells. In the double labeling experiments, GluR6 and KA1 subunits of GluRs were mainly expressed with Gus. These results suggest that kainate glutamate receptors preferentially expressed in type II taste bud cells in rat.

• Journal of Microbiology
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• 제43권4호
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• pp.361-365
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• 2005

In this study, we chronicle the establishment of a novel transformation system for the unicellular marine green alga, Dunaliella salina. We introduced the CaMV35S promoter-GUS construct into D. salina with a PDS1000/He micro-particle bombardment system. Forty eight h after transformation, via histochemical staining, we observed the transient expression of GUS in D. salina cells which had been bombarded under rupture-disc pressures of 450 psi and 900 psi. We observed no GUS activity in either the negative or the blank controls. Our findings indicated that the micro-particle bombardment method constituted a feasible approach to the genetic transformation of D. salina. We also conducted tests of the cells' sensitivity to seven antibiotics and one herbicide, and our results suggested that 20 ${\mu}g$/ ml of Basta could inhibit cell growth completely. The bar gene, which encodes for phosphinothricin acetyltransferase and confers herbicide tolerance, was introduced into the cells via the above established method. The results of PCR and PCR-Southern blot analyses indicated that the gene was successfully integrated into the genome of the transformants.

• 생명과학회지
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• 제19권3호
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• pp.378-383
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• 2009

본 연구는 Agrobacterium을 이용한 효율적인 호접란 형질 전환 시스템의 확립을 위해 실시하였고, 호접란 PLB의 대량증식을 위한 배지 조성은 하이포넥스 기본 배지에 활성탄 1g/l, 티아민 0.1 mg/l 및 sucrose 30 g/l을 배지에 첨가한 경우 PLB가 안정적으로 증식되었다. 계대 배양시 PLB 절단 방법이 PLB의 생존과 증식에 큰 영향을 미치며, PLB 위쪽으로 부터 1/3 부위를 절단하여 계대 배양한 경우 90% 이상의 가장 높은 증식률을 나타났다. Agrobacterium 접종 시 균이 묻어 있는 침으로 PLB를 찔러 상처를 내는 dipping 방법을 이용하여 형질전환 효율을 높일 수 있었다. Agrobacterium 배양액의 흡광도 값이 0.8일 때 형질전환 효율이 가장 높았고, 형질전환된 PLB의 선발은 1 mg/l 정도의 저농도 hygromycin을 함유한 배지에 계대배양 하는 것이 효율적이었다. 호접란 형질전환체를 먼저 GUS assay를 통하여 선발하였고, 선발된 개체로부터 genomic DNA를 추출하여 GUS 특이적 primer와 probe로 PCR과 Southern blot 분석을 수행하고 유전자의 도입여부를 조사한 결과 GUS 염색된 형질전환체에서 정상적으로 GUS 유전자가 도입된 것을 확인할 수 있었다.

• 한국산림과학회지
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• 제87권3호
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• pp.334-340
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• 1998

이 연구(硏究)는 promoter가 없는 외래(外來) 유전자(遺傳子)를 양황철의 genome에 인위적으로 삽입시킨 후 도입된 유전자(遺傳子)가 식물 프로모터의 영향으로 발현되는 현상을 이용하여 식물의 프로모터 혹은 유전자(遺傳子) 발현조절 염기서열(鹽基序列)을 분리, 구명하기 위해 수행되었다. 형질전환된 세포의 선발을 위하여 nptII 유전자(遺傳子)를 선발 표지로 사용하였고, 발현되는 유전자(遺傳子)의 검정을 위한 reporter보는 GUS 유전자(遺傳子)를 사용하였다. 형질전환 후 재분화된 3클론 중 nptII 및 GUS의 발현에 모두 양성인 개체의 DNA에서 730bp 염기서열(鹽基序列)을 inverse PCR로 증폭 분리하여 클로닝하고 이의 염기서열(鹽基序列)을 구명하였다. 이 염기서열(鹽基序列)은 Eucalyptus gunnii의 CAD(Cinnamyl Alcohol Dehydrogenase) 유전자(遺傳子)와 전체적으로 약 88%의 상동성(相同性)을 보였다. 이 결과에 의하면 inverse PCR로 증폭된 부분은 포플러의 CAD 유전자(遺傳子)의 일부를 포함한 조절인자로 생각된다. 이렇게 클로닝된 DNA 염기서열(鹽基序列)과 GUS fusion된 합성 DNA를 particle bombardment 법을 이용하여 포플러 잎에 도입시킨 결과, 청색반점(靑色斑點)이 생성되는 것으로 보아, 분리된 부위가 식물체내에서 발현조절기능을 하는 일부분으로 작용하는 것으로 생각된다.

• Plant Biotechnology Reports
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• 제3권4호
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• pp.325-331
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• 2009

To efficiently express a gene of interest in transgenic plants, the choice of promoter is a crucial factor as it directly affects the expression of the transgene that will yield the desired phenotype. The Arabidopsis ${\beta}-carotene$ hydroxylase 1 gene (AtBch1) shows constitutive and ubiquitous expression and was thus selected as one of best candidates for constitutive promoter analysis by both in silico northern blotting and semi-quantitative RT-PCR analysis. To investigate AtBch1 promoter activity, the 1,981-bp 5'-upstream region of this gene was fused with ${\beta}-glucuronidase$ (GUS) and transformed into Arabidopsis. Through the molecular characterization of transgenic leaf tissues, the AtBch1 promoter generated strong activity that drives 1.8- and 2-fold higher GUS expression than the cauliflower mosaic virus 35S (35S) promoter at the transcriptional and translational levels, respectively. Furthermore, the GUS enzyme activity driven by the AtBch1 promoter was 2.8-fold higher than that produced by the 35S promoter. By histochemical GUS staining, the ubiquitous expression of the AtBch1 promoter was observed in all tissues of Arabidopsis. Semi-quantitative RT-PCR analysis with different tissues further showed that this promoter serves as a strong constitutive driver of transgene expression in dicot plants.

• Journal of Ginseng Research
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• 제41권3호
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• pp.419-427
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• 2017

Background: Glycosylation of natural compounds increases the diversity of secondary metabolites. Glycosylation steps are implicated not only in plant growth and development, but also in plant defense responses. Although the activities of uridine-dependent glycosyltransferases (UGTs) have long been recognized, and genes encoding them in several higher plants have been identified, the specific functions of UGTs in planta remain largely unknown. Methods: Spatial and temporal patterns of gene expression were analyzed by quantitative reverse transcription (qRT)-polymerase chain reaction (PCR) and GUS histochemical assay. In planta transformation in heterologous Arabidopsis was generated by floral dipping using Agrobacterium tumefaciens (C58C1). Protein localization was analyzed by confocal microscopy via fluorescent protein tagging. Results: PgUGT72AL1 was highly expressed in the rhizome, upper root, and youngest leaf compared with the other organs. GUS staining of the promoter: GUS fusion revealed high expression in different organs, including axillary leaf branch. Overexpression of PgUGT72AL1 resulted in a fused organ in the axillary leaf branch. Conclusion: PgUGT72AL1, which is phylogenetically close to PgUGT71A27, is involved in the production of ginsenoside compound K. Considering that compound K is not reported in raw ginseng material, further characterization of this gene may shed light on the biological function of ginsenosides in ginseng plant growth and development. The organ fusion phenotype could be caused by the defective growth of cells in the boundary region, commonly regulated by phytohormones such as auxins or brassinosteroids, and requires further analysis.