• 제목/요약/키워드: GUS expression

검색결과 160건 처리시간 0.022초

Acquirement of transgenic rose plants from embryogenic calluses via Agrobacterium tumefaciens (배발생 캘러스를 이용한 아그로박테리움 매개형질전환 장미 식물체 획득)

  • Lee, Su-Young;Lee, Jung-Lim;Kim, Won-Hee;Kim, Seung-Tae;Lee, Eun-Kyung
    • Journal of Plant Biotechnology
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    • 제37권4호
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    • pp.511-516
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    • 2010
  • The process to acquire intron-GUS gene-expressed transformants from somatic embryos (including embryogenic calli) of Rosa hybrida cv. 'Sweet Yellow' using Agrobacterium-meditated transformation method was reported in this study. Somatic embryos including embryogenic calluses were infected with Agrobacterium tumefaciens AGL1 strain (O.D = 0.7~1.6) including intron-GUS gene for 30 min, and were co-cultured for 3 days. After co-cultivation, they were cultured on embryo germination medium (EGM) supplemented with $250\;mg{\cdot}L^{-1}$ cefotaxim at $4^{\circ}C$ for 7 days. Then, transient GUS gene expression was observed. Shoots were regenerated from the shoot primodia induced from the intron-GUS gene-transferred either somatic embryos or embryogenic calli cultured on EGM supplemented with both cefotaxim $250\;mg{\cdot}L^{-1}$ and ppt $2\;mg{\cdot}L^{-1}$. Before induction of rooting from shoots cultured on shoot growing medium supplemented with both cefotaxim $250\;mg{\cdot}L^{-1}$ and ppt $2\;mg{\cdot}L^{-1}$, the shoots were cultured on multi-shoot induction medium supplemented with both cefotaxim $250\;mg{\cdot}L^{-1}$ and ppt $2\;mg{\cdot}L^{-1}$ to induce multi-shoots. When expression of the gene from a part of the multi-shoots was identified by GUS transient assay, the putative transgenic multishoots were transferred to rooting medium supplemented with cefotaxim $250\;mg{\cdot}L^{-1}$. After the formation of healthy roots, transgenic plantlets were transferred to the greenhouse after acclimatization. The expression rate of the intron-GUS gene in the multi-shoots was 100%.

Efficient Transformation of Trifolium repens L. Using Acetosyringone (Acetosyringone을 이용한 효율적인 White Clover의 형질전환)

  • TaeHoKwon
    • Korean Journal of Plant Resources
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    • 제10권2호
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    • pp.107-113
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    • 1997
  • Transformants of White Clover(Trifolium repens L.) were efficiently produced from immature seed derived callus cocultivated with Agrobacterium twnefaciens LBA4404 harboring plant binary vector. pBI121, using acetosyringone. The mean frequencies of transformants on the two kanamycin-containing media were 16 to 19% when the immature seed-derived calli were infected with bacteria cultured in the presence of 100$\mu$M acetosyringone compared with 7% in media without acetosyringone. Transgenic white clover was subject to molecular analysis for integration into plant nuclear genome and expression of $\beta$-glucuronidase(GUS) gene. PCR and Northern blot analyses demonstrated that GUS gene was integrated into white clover nuclear genome and expressed into its mRNA. The expression of GUS gene into its protein was confirmed by spectrophotometric assay of GUS activity.

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Expression of gus and gfp Genes in Ggrlic (Allium sativum L.) Cells Following Particle Bombardment Transformation

  • Lacorte, Cristiano;Barros, Daniella
    • Journal of Plant Biotechnology
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    • 제2권3호
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    • pp.135-142
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    • 2000
  • The activity of promoter sequences was evaluated in garlic cells using the $\beta$-glucuronidase (GUS) gene as a reporter. Histochemical GUS assay indicated transient GUS activity in leaf, callus and root cells 48 hours after particle bombardment transformation. Quantitative fluorometric assays in extracts of transformed leaves demonstrated that the CsVMV promoter induced the highest level of gene expression, which was, on average, ten fold the level induced by CaMV35S and by the Arabidopsis Act2 promoters and two fold the level expression observed with a construct containing a double CaMV35S plus the untranslated leader sequence from AMV. No activity or very low levels were observed when cells were transformed with plasmids rontaining the typical monocot promoters, Actl, from rice or the Ubi-1, from maize. The green fluorescent protein (GFP) was also tested as a marker gene for garlic transformation. Intense fluorescence was observed in leaf, callus and root cells transformed with a construct containing the gfp gene under control of the CaMV35 Promoter. No fluorescence was detected when the gfp was under control of the Ubi-1 promoter.

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Transformation of Cell Wall-weakened Perilla Seedlings Using Phenolic Compound-treated Agrobacterium Cells and Recombinant Protein Expression (페놀화합물 처리 Agrobacterium 및 세포벽 약화 들깨새싹을 이용한 형질전환과 재조합 단백질 발현)

  • Chung, Il-Kyung;Shin, Dong-Il;Park, Hee-Sung
    • KSBB Journal
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    • 제24권6호
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    • pp.598-601
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    • 2009
  • Perilla [Perilla frutescens (L.) Britt] seedlings are easy to grow and eaten as the health vegetable sprout. Two day old perilla seedlings since germination were given a mild wounding using cell wall lytic NaOH/SDS solution for infiltration with recombinant Agrobacterium cells treated with phenolic compounds. In the analysis of fluorometric GUS gene expression for the transformed perilla seedlings, GUS enzyme activity was the highest by the combined treatments of 50 mM acetosyringone and 0.5% NaOH solution containing 0.01% SDS implying a synergic effect. This result could be successfully applied for demonstrating hepatitis B virus antigen (HBsAg) protein expression.

Development of Transient Expression System Using Transformed Seedlings of Brassica napus var. napus (유채유묘의 형질전환을 통한 일시발현시스템의 개발)

  • Shin, Dong-Il;Park, Hee-Sung
    • KSBB Journal
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    • 제21권6호
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    • pp.489-492
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    • 2006
  • For molecular breeding purpose, genetic transformation of Brassica napus cultivars has been extensively performed using Agrobacterium method. B. napus cv. napus, one of major oil crops, can be transformed via Agrobacterium-based method. We demonstrated that Agrobacterium-mediated transformation via vacuum infiltration slightly worked for the seedlings of B. napus cv. napus according to fluorometric GUS enzyme analysis. In contrast, transformation efficiency was highly enhanced when the seedlings, prior to agroinfiltration, were treated with sodium hydrosulfite solution as a chemical wounding agent. GUS gene expression in transformed seedlings that was confirmed by RT-PCR suggests their usefulness for the development of transient expression system.

Functional Characterization of NtCDPK1 in Tobacco

  • Lee, Sang Sook;Yoon, Gyeong Mee;Rho, Eun Jung;Moon, Eunpyo;Pai, Hyun-Sook
    • Molecules and Cells
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    • 제21권1호
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    • pp.141-146
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    • 2006
  • We previously showed that NtCDPK1, a tobacco calcium-dependent protein kinase, interacts with and phosphorylates the Rpn3 regulatory subunit of the 26S proteasome, and that both NtCDPK1 and Rpn3 are mainly expressed in rapidly proliferating tissues, including shoot and root meristem. In this study, we examined NtCDPK1 expression in roots using GUS expression in transgenic Arabidopsis plants, and investigated its function in root development by generating transgenic tobacco plants carrying a sense NtCDPK1 transgene. GUS activity was first detected in roots two days after sowing. In later stages, strong GUS expression was detected in the root meristem and elongation zone, as well as the initiation sites and branch points of lateral roots. Transgenic tobacco plants in which NtCDPK1 expression was suppressed were smaller, and their root development was abnormal, with reduced lateral root formation and less elongation. These results suggest that NtCDPK1 plays a role in a signaling pathway regulating root development in tobacco.

Introduction and Expression of Foreign Genes in Rice Cells by Particle Bombardment

  • Jeon, Jong-Seong;Jung, Hou-Sung;Sung, Soon-Kee;Lee, Jong-Seob;Choi, Yang-Do;Kim, Han-Jip;Lee, Kwang-Woong
    • Journal of Plant Biology
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    • 제37권1호
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    • pp.27-36
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    • 1994
  • For establishing a transformation system of rice, an efficient introduction of foreign genes into embryogenic cell suspension by particle bombardment was conducted. The particle inflow gun based on the acceleration of DNA-coated tungsten particles using pressurized helium was constructed for delivery of DNA into rice cells. Several bombardment parameters were optimized using the transient expression of GUS gene. The conditions that gave the highest GUS gene expression of about 1000 blue spots per g fresh weight of bombarded cells include treatment of the cells with 0.5 M osmotic pressure, and use of the 410 kPa helium, 110 mm target distance, 13 mm syringe filter holder and 5 $\mu$L DNA/tungsten mixtures. It was also confirmed that rice actin promoter-intron construct gave the highest expression of all promoter-sequences studied. Eight weeks after the bombardment, stably transformed calluses were obtained on the selection medium containing 100 mg/L G418 and showed the strong activity in in situ GUS assay.

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Impact of Physical, Chemical and Biological Factors on Lily (Lilium longiflorum cv. Georgia) Pollen Growth and GUS Expression Via Agro-infiltration (물리적, 화학적, 생물적 요인에 의한 백합 (Lilium longiflorum cv. Georgia) 화분의 생장 및 Agro-Infiltration을 이용한 GUS 발현)

  • Park, Hee-Sung
    • Journal of Plant Biotechnology
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    • 제31권4호
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    • pp.279-283
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    • 2004
  • To lily (Lilium longflorum cv. Georgia) pollen, impacts by some physical, chemical and biological factors were examined in respects of its growth and transient gene expression via agro-infiltration. Rolling movement in liquid medium or vacuum pressure during Agro-infiltration was regarded as a impact that should be minimized for normal pollen growth. Pollen growth was maintained well in relatively broad range of temperature (19 to 27$^{\circ}C$) or pH (5.0 to 8.0). Chemical factors such as cefotaxime (up to 300mg/L), acetosyringone (up to 800 $\mu$M) and syringealdehyde (up to 800 $\mu$M) did not show any harmful effects but kanamycin severely did even at concentration as low as 25mg/L in some cases. For GUS gene expression, acetosyringone at 200 to 400 $\mu$M slightly improved the efficiency while syringealdehyde did not. Brief agro-infiltration followed by 18 hr of co-incubation of pollen along with Agrobacterium was suggested as a condition basically required for the transient expression system using lily pollen regardless of the presence of acetosyringone.

Activation of Barley S-Adenosylmethionine Synthetase1 Gene Promoter in Response to Phytohormones and Abiotic Stresses

  • Kim, Jae-Yoon;Kim, Dae-Yeon;Jung, Je-Hyeong;Hong, Min-Jeong;Heo, Hwa-Young;Johnson, Jerry W.;Kim, Tae-Ho;Seo, Yong-Weon
    • Journal of Crop Science and Biotechnology
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    • 제10권1호
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    • pp.50-56
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    • 2007
  • Barley S-adenosylmethionine synthetase1 gene, which was differentially expressed in seed development of extra early barley, was regulated by the phytohormones and abiotic stresses. In order to identify the regulation regions which were involved in transcriptional control of the phytohormones and abiotic stresses, we isolated 1459 bp fragment of HvSAMS1 gene promoter using genome walking strategy and deletion series were constructed. Deleted upstream fragments(-1459, -1223, -999, -766, -545, -301 bp) were fused to the GUS reporter gene and evaluated via Agrobacterium-mediated transient expression assay. Increased GUS activity of HvSMAS1 promoter -301/GUS construct under each of NaCl, $GA_3$, ABA and ethylene application was found. However, GUS activity was negligible in the leaves transformed with the HvSMAS1 promoter(-1459, -1223, -999, -766 and -545)/GUS constructs. No significant induction of GUS activity was observed for the ethionine and spermidine treatments. In order to locate promoter sequence of the HvSAMS1 gene that was critical for the activation of gene expression, deletion and addition promoter derivatives(+, includes 43 bp of 5' ORF) of the HvSAMS1 gene fused to the GUS reporter gene were applied. The tobacco leaves which harbored the additional HvSAMS1 promoter(-1459+, -1459 to -546, -545+ and -301+)/GUS construct did not significantly induce GUS activity as compared to the HvSAMS1 promoter(-1459, -545 and -301)/GUS constructs under each of NaCl, ABA and $GA_3$ treatment. However, the GUS activity was high in the tobacco leaves which harboring the -211 to -141 regions of the HvSAMS1 promoter. This result suggested that HvSAMS1 gene expression might be regulated by this region(from -211 to -141).

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Lily Pollen Growth in vitro and Agrobacterium-mediated GUS Gene Transformation via Vacuum-Infiltration

  • Park, In-Hae;Park, Hee-Sung
    • 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.