• 제목/요약/키워드: gusA

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Expression and Inheritance Patterns of Gus Gene Driven by an Endosperm-Specific Promoter in Transgenic Tobacco (배유 특이 프로모터에 의해 유도된 GUS 유전자의 형질전환 담배 내에서의 발현 및 유전 양상)

  • Park, Young Doo;Kim, Hyoung Seok
    • Horticultural Science & Technology
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    • v.18 no.5
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    • pp.594-598
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    • 2000
  • This study was carried out to investigate the tissue-specific expression of ${\beta}$-glucuronidase (gus) gene driven by endosperm-specific promoter (Z4 promoter) in the transgenic tobacco and to find out inheritance pattern of transgene to the next generation. Tobacco (Nicotiana tabaccum cv. Havana SR1) was transformed with Agrobacterium tumerfaciens LBA4404 harboring BV3 construct containing gus gene driven by Z4 promoter and a kanamycin resistant gene. Seven hundred bp PCR products, indicating the presence of npt II gene, were found in the all eight transformants by PCR analysis using nptII primers. To study the expression pattern of the two different kind of promoters, leaf disks of the Z4pro-gus-transformed plants and 35Spro-gus-transformed plants were analyzed histochemically for gus activity. As a result, leaf disks of Z4pro-gus-transformed plants showed very weak and partial positive gus activity. In contrast, leaf disks of 35Spro-gus-transformed plants showed relatively strong positive gus activity. To investigate the expressed position of Z4 promoter, seeds from Z4pro-gus-transformed plants and 35Spro-gus-transformed plants were analyzed histochemically for gus activity. Z4pro-gus-transformed seeds showed positive gus activity restricted to the endosperm. However, the blue-colored product in 35Spro-gus-transformed seeds was observed in all the area including endosperm. Kanamycin resistance assay showed that transgenes were stably inherited to next generation in all lines.

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Development of a Screening System for Plant Defense-Inducing Agent using Transgenic Tobacco Plant with PR-1a Promoter and GUS Gene

  • Oh, Sang-Keun;Lee, Seon-Woo;Kwon, Suk-Yoon;Choi, Do-Il
    • The Plant Pathology Journal
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    • v.21 no.3
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    • pp.288-292
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    • 2005
  • Pathogenesis-related protein-1a (PR-1a) is strongly induced in tobacco plants by pathogen attack, exogenous salicylic acid (SA) application and by other developmental processes. In order to develop a rapid screening system for the selection of plant defense-inducing compounds originated from various sources, we have transformed tobacco Samsun NN plants with a chimeric construct consisting of GUS $(\beta-glucuronidase)$. In the $T_1$ generation, three transgenic lines having stable GUS expression were selected for further promoter analysis. Using GUS histochemical assay, we observed strong GUS induction driven by PR-1a promoter in PR1a-GUS transgenic tobacco leaves in response to the exogenous application of SA or benzol (1,2,3) thiadiazole-7-carbothioic acid S-methyl ester (BTH), a SA­derivative compound. In addition, GUS expression was maintained locally or systemically in PR1a-GUS transgenic line $\#5\;T_2$ generation) until after 3 days when they were treated with same chemicals. Our results suggested that the PR1a-GUS reporter gene system in tobacco plants may be applicable for the large-scale screening of defense-inducing substances.

The increased GUS gene inactivation over generation in Arabidopsis transgenic lines (애기장대 형질전환 식물체의 세대경과에 따른 GUS유전자의 비활성화에 관한 연구)

  • Park, Soon-Ki
    • Journal of Life Science
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    • v.12 no.1
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    • pp.67-76
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    • 2002
  • The effect of transgene inactivation in T2, T3 and F2 generations was analyzed in progeny seedlings which had been generated by Agrobacterium (LBA4404/pBI121)-mediated transformation in Arabidopsis thaliana. In a system which investigated in the expression of $\beta$-glucuronidase(GUS)gene in kanamycin-resistant (ke $n^{R}$)seedlings, GUS inactivated seedlings were observed in 5 of 12 tested lines of T2 generation and the frequency of GUS inactivation was approximately 2.3%. Lines with multi-copies of T-DNA exhibited severe GUS gene inactivation with the frequency of 5.8% in T2 generation. In T3 generation lines exhibited GUS gene inactivation with the frequency of 1.3%. In contrast, inactivation increased dramatically up to 12.6% in multi-copy T-DNA line. A similar phenomenon was also found in F2 progeny from a transgenic line which had been crossed with wild-type Arabidopsis plant, WS-O (GUS gene inactivation frequency 9.9%). These results indicate that the foreign gene introduced into the plant was inactivated progressively in its transmission during subsequent generations and the transgenic line with multi-copies of T-DNA tended to show more increased inactivation.

Tissue Specific Expression of Wound-Inducible RCaM-2 Promoter in Transgenic Tobacco Plants (상처에 의해서 유도되는 벼 calmodulin promoter의 transgenic 담배에서조직 특이적 발현)

  • Choi Young Ju
    • Journal of Life Science
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    • v.15 no.2 s.69
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    • pp.176-181
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    • 2005
  • To study calmodulin (CaM) gene expression and its regulation, rice CaM promoter (RCaM-2) was isolated and fused to $\beta-glucuronidase$ (GUS), reporter gene. X-Glue staining patterns revealed that GUS localization is high in meristemic tissues such as the stem apex, stolen tip, and vascular regions. GUS staining in the transverse sections of stem and petiole was restricted to the inside of the vascular system, and cortex and epidermis located outside of the vascular system usually did not show GUS staining even a plant that expressed strong activity. GUS activity was found to be tissue specific expressed and exhibited a dramatic transient increase in response to wounding. These results suggest that the 5'-flanking region of RCaM gene regulates wound-inducible expression.

Transformation of Plant Cells by Gene Transfer : Construction of a Chimeric Gene Containing Deleted Maize Alcohol Dehydrogenase Intron and ${\beta}-Glucuronidase$ Gene and Its Expression in Potato (유전자 도입에 의한 식물세포의 형질전환 : 옥수수 알코올 탈수소효소 유전자의 절단된 인트론 및 ${\beta}-Glucuronidase$ 유전자를 함유하는 키메라 유전자의 제조와 감자에서의 발현)

  • 이광웅
    • Journal of Plant Biology
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    • v.35 no.3
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    • pp.237-245
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    • 1992
  • To understand the properties of the cauliflower mosaic virus (CaMV) 35S promoter and the effect of the deleted maize alcohol dehydrogenase I-S (Adhl-S) intron 1 on the expression of the CaMV $35S{\beta}-glucuronidase$ (GUS) gene in potato (Solanum tuberosum L. cv. Superior), we constructed a chimeric gene and transferred it into potato with Agrobacterium tumefaciens mediated method. The pLS201, a gene transfer vector of 17.7 kilobase pairs, was composed of the CaMV 35S promoter, the 249 base pairs of deleted maize Adhl-S intron 1, the GUS reporter gene, and the kanamycin resistance gene as a selectable marker for transformation. The GUS activity was examined by histochemical and spectrophotometric assay in transformed potato plants. The GUS activity was found primarily around the vascular tissue cells in stem and root. In the spectorophotometric assay, the level of GUS activity of transgenic potato transformed with CaMV 35S/249 bp of intron 1 fragment-GUS (pLS201) was compared with that of potato transformed with CaMV 35S-GUS (pBI121). The quantitative spectrophotometric assay showed that the level of GUS activity in potato transformed with pLS201 was higher in leaf, stem and root by 30-, 34- and 42-fold, respectively than those in potato transformed with pBI121. This results indicate that the inclusion of the deleted maize Adhl-S intron 1 resulted in increament of the GUS gene expression in transgenic potato.potato.

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Expression of $\beta$-Glucuronidase (GUS) Gene in Transgenic Lettuce (Lactuca sativa L.) and Its Progeny Analysis (형질전환된 상추내에서 GUS 유전자의 발현 및 후대검정)

  • CHUNG, Jae Dong;KIM, Chang Kil;KIM, Kyung Min
    • Korean Journal of Plant Tissue Culture
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    • v.25 no.4
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    • pp.225-229
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    • 1998
  • Agrobacterium tumefaciens LBA 4404 harboring binary vector pBI 121 was used for genetic transformation of lettuce(Lactuca Sativa L.). Optimal shoot regeneration from cotyledon explants was obtained in MS medium supplemented with 0.1mg/L NAA and 1.0 mg/L 2ip. In this condition, cotyledon explants were cocultivated with A, tumefaciens for 2 days, and then transferred to selection medium supplemented with 50 mg/L kanamycin and 500 mg/L carbenicillin. These explants were subsequently subcultured every 2 weeks on shoot induction medium. PCR analysis indicated that the GUS gene was stably integrated into the nuclear genome of lettuce. Histochemical analysis based on the enzymatic activity of the CUS protein showed that GUS activity was associated with vascular tissue in leaves and roots. Progenies of Ro plants demonstrated a linked monogenic segregation for GUS gene.

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Development of an Agrobacterium-mediated Transient Expression System for Intact Leaves of Chili Pepper (Agrobacterium을 이용한 고추의 Transient Expression 시스템)

  • Seong, Eun-Soo;Joung, Young-Hee;Choi, Doil
    • Journal of Plant Biotechnology
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    • v.31 no.3
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    • pp.185-190
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    • 2004
  • We established a transient gene expression system in chili pepper leaves based on Agrobacterium-mediated transformation of GUS gene. For the best GUS transient expression, two step culture system was adopted. When the Agrobacterium tumefaciens cell density of pre-culture was $A_{600nm}$ 0.3, the cells were harvested and diluted to $A_{600nm}$ 0.8 with virulence induction medium after cell harvested. The addition of acetosyringone (200 $\mu$M) in virulence induction step was a key factor for successful transient expression. Additionally, Younger leaves showed more effective transient expression than older leaves. Temporally, the strongest intensity of GUS expression was detected at 2 days after infiltration. These results demonstrate that Agrobacterium-mediated transient expression can be used for a simple in vivo assays of plant promoters, transcription factors and furthermore provide efficient protocol for chili pepper transformation.

Genetic Transformation of Lettuce (Lactuca sativa L.) with Agrobacterium tumefaciens (Agrobacterium tumefaciens에 의한 상추 (Lactuca sativa L.)의 형질전환)

  • 최언옥;양문식;김미선;은종선;김경식
    • Korean Journal of Plant Tissue Culture
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    • v.21 no.1
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    • pp.55-58
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    • 1994
  • Agrobacterium tumefaciens LABA4404 harboring plant binary vector, pBI121, was used for genetic transformation of lettuce (Lactuca sativa t.). Cotyledon segments were infected with A. tumefaciens LBA4404 by cocultivation method and regenerated. Regenerated letture was subject to molecular analyses for integration into plant nuclear genome and expression of ${\beta}$-glucumnidase (GUS) gene. Southern and Northern blot analyses demonstrated that GUS gene was integrated into plant nuclear genome and expressed into its mRNA. The expression of GUS gene into its protein was confirmed by specetrophotometric assay of GUS activity.

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Electroporation Conditions for DNA Transfer into Somatic Embryogenic Cells of Zoysia japonica (들잔디 체세포 배발생 세포로의 DNA 전입을 위한 Electroporation 조건 구명)

  • 박건환;안병준
    • Korean Journal of Plant Tissue Culture
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    • v.25 no.1
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    • pp.13-19
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    • 1998
  • We have reported previously that intact embryogenic cells can be used instead of protoplasts for electroporation-mediated transformation of zoysiagrass and rice. In this study, conditions of the tissue electroporation were examined to optimize the procedures. Embryogenic cell suspensions were established in liquid MS medium containing 2 mg/L of 2,4-D with embryogenic calluses induced from mature embryos of Z. japonica. The suspension-cultured cell clumps were electroporated with 35S-gusA expression vector DNA, and degrees of DNA introduction into the cells were determined by histological expression rates of the gusA marker gene. DNA transfer into the cell clumps occurred in wide range of voltage (100-400 V) and capacitance (10-1980 $\mu\textrm{F}$), but more in the ranges of 200-300 V and 330-800 $\mu\textrm{F}$ DNA concentrations higher than 6 $\mu\textrm{g}$/mL were adequate for GUS expression of the electroporated cells. DNA transfers were confirmed in all three embryogenic cell lines but only in one out of eleven non-embryogenic lines. Positive GUS expressions occurred with DNAs added even 20-40 h after pulse treatments. As a promoter of gusA, Act1 and Ubi1 were effective 7 and 5 times than 35S respectively in number of GUS expression units on electroporated cell clumps. Embryogenic cell clumps survived and regenerated into plantlets after pulse treatments of wide range of conditions.

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Action mechanism of upstream open reading frame from S-adenosylmethionine decarboxylase gene as a in vivo translational inhibitor (S-Adenosylmethionine decarboxylase 유전자의 upstream open reading frame이 in vivo에서 translational inhibitor 로서의 작용 기작)

  • Choi, Yu-Jin;Park, Ky-Young
    • Journal of Plant Biotechnology
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    • v.38 no.1
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    • pp.87-93
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    • 2011
  • S-Adenosylmethionine decarboxylase (SAMDC; EC 4.1.4.50), a key enzyme for polyamines biosynthesis, was tightly regulated for homeostatic levels. Carnation SAMDC gene (CSDC9) has an small upstream open reading frame (uORF) of 54 amino acids in 5'-leader sequence. To explore the functional mechanism of uORFs in controlling translation, we used a GUS reporter gene driven with the 35S promoter and uORF region of SAMDC gene for making transgenic tobacco plants. In our experiment, there were a translational inhibition of its downstream GUS ORF by SAMDC uORF sequence or SAMDC uORF protein. Expecially, translational inhibition was most effective in point-mutated construct, in which the start codon was changed. Therefore, this results suggested the ribosomal stalling might be involved in this translational inhibitory process. The frame shift in amino acid sequence of SAMDC uORF with start codon and stop codon resulted in a moderate increasing in GUS activity, suggesting the native amino acid sequence was important for a function as a translational inhibitor. Also, we showed that the production of GUS protein was significantly inhibited in the presence of the small uORF using histochemical analysis of GUS expression in seedlings and tobacco flowers. Importantly, the small uORF sequence induced a real peptide of 5.7 kDa, which was provided the presence of SAMDC uORF peptide band using an in vitro transcription/translation system. The peptide product of uORF might interact with other components of translational machinery as well as polyamines, which was resulted from that polyamine treatment was inhibited GUS protein band in SDS-PAGE experiment.