• Journal of Microbiology and Biotechnology
• /
• 제18권4호
• /
• pp.754-758
• /
• 2008

Agrobacterium tumefaciens-mediated transformation (ATMT) was successfully applied to Monascus ruber. The optimum cocultivation time was 84 h with an efficiency of 900 to 1,000 transformants when $1{\times}10^6$ spores were used with the same volume of bacteria. The stability of transform ants was over 98% after five generations. When M. ruber was transformed with A. tumefaciens YL-63 containing the green fluorescent protein gene (egfp), the green fluorescent signal was observed throughout hyphae, confirming expression of the gene. This efficient transformation and expression system of M. ruber by ATMT will facilitate the study of this fungus at a molecular genetic level.

• 생약학회지
• /
• 제23권3호
• /
• pp.137-141
• /
• 1992

The cells of Rubia cordifolia var. pratensis were transformed by Agrobactrium tumefaciens strain 11157. Surface-sterilized young leaves and stems of the plants were cocultivated with bacterial suspensions. Crown galls induced from stems were cultured with variation of culturing conditions and compared with untransformed cells. The growth rates and production of anthraquinone pigments of cells were remarkably improved by transformation. Furthermore, hairy roots were induced by inoculation or cocultivation with Agrobacterium rhizogenes strains.

• Journal of Plant Biology
• /
• 제38권4호
• /
• pp.365-371
• /
• 1995

Binary vectors, pBI-ActR1, pBI-ActF1 and pBSH-ActR1, were constructed using pGA642, the replication origin of pTi12 and the rice actin promoter. The sizes of pBI-ActR1, pBI-ActF1 and pBSH-ActR1 were 12.9 kb, 13.2 kb and 11.95 kb, respectively. These vectors containing a rice actin promoter followed by a GUS structural gene could induce stronly the expression of GUS gene in transformed rice cells. Rice explants from 3-4 day old seedlings after germinatin were cocultured with A. tumefaceins harboring pBI-ActR1, pBI-ActF1 or pBSH-ActR1, and then GUS expression in the explants was assayed. Transformation of rice explants by these binary vectors was tissue-specific, such that the meristematic regions of shoot apex, root and hypocotyl were transformed by these binary vectors.

• Journal of Plant Biotechnology
• /
• 제35권4호
• /
• pp.337-343
• /
• 2008

We generated transgenic tomato plants with Arabidopsis thaliana $H^+$/cation exchanger gene (C4X4) by Agrobactrium-mediated transformation. We confirmed transgene copy number and transcription by Southern and Northern blot analyses. The intact CAX4-expressing tomato (Lycopersicon esculentum) fruits contained 63-71% more calcium ($Ca^{2+}$) than wild-type fruits. Moreover, ectopic expression of C4X4 in tomato fruits did not show any significant increase of the four kinds of metallic cations analyzed ($Mg^{2+}$, $Fe^{2+}$, $Mn^{2+}$, and $Cu^{2+})$. The C4X4-expressing tomato plants including their fruits did not show any morphological alternations during whole growth period. These results suggest the enhanced Ca-substrate specificity of CAX4 exchanger in tomato. Therefore, intact CAX4 exchanger can be a useful tool for $Ca^{2+}$ nutrient enrichment of tomato fruits with reduced accumulation of undesirable cations.

• KSBB Journal
• /
• 제16권5호
• /
• pp.523-525
• /
• 2001

커들란은 용액의 pH 7.0에서 salt 상태로 존재하기 때문에 커들란만이 액상에 녹아 있는 경우에는 분리공정은 원심분리를 함으로써 분리가 가능하다. 그러나, 세포외 다당류로 생산된 커들란읜 세포, 염 의 무기물이 섞여 있기 때문에 이러한 물질을 제거하는 것이 커들란 분리의 중요한 변수가 되었다. 커들란의 생산을 위한 분리에 관한 보고는 원심분리 방법을 이용한 것으로 알려져 있음 세포 및 불순물 등을 제거하여 순수한 커들란을 분리하기 위해 pH swing separation (pH가 높은 경우 커들란이 용액에 녹는 성질을 이용)을 이용하였으나 원심분리를 수 차례 반복해야 되기 때문에 소요되는 동력이 많이 필요하여 에너지 절감형 및 오염절감형의 새로운 공정의 개발이 필요하게 되었다. 온 연구에서는 pH swing separation 에서 동력비가 많은 드는 단점을 감소시키기위하여 커들란의 물리 화학적인 특성을 고려한 분리공정을 개발하였다. 향후 공정 개선을 위한 지속적인 연구가 필요하며 완성시 경제적이 공정을 개발하는 것이 가능할 것이다.

• The Plant Pathology Journal
• /
• 제24권3호
• /
• pp.296-304
• /
• 2008

The transient and rapid expression system of a foreign protein in planta is a very useful technique in biotechnology application. We have investigated optimum condition of Agrobacterium-infiltration technique in which expression level of foreign proteins were maximized without detrimental effects on plants using GFP and Cucumber mosaic virus 2b protein, which is known as an enhancer of gene expression and a suppressor of post-transcriptional gene silencing(PTGS). The optimum expression level of both RNA and protein of GFP with minimum leaf impairment was obtained at $OD_{600}$=0.2 of Agrobactrium inocula. The steady-state levels of GFP RNA and protein generally peaked at 3 and 7 days post-infiltration(dpi), respectively. In the presence of 2b, both the magnitude and duration of GFP expression was highly increased and we could detect GFP level until 17 dpi. On the other hands, the 2b-mediated higher accumulation of foreign proteins resulted in the repression of normal leaf growth, possibly due to the limitation of supply of energy or materials required for growth maintenance. Using this Agrobacterium-infiltration system with 2b and GFP, we tested a hypothesis for the threshold model of PTGS initiation. Four GFP transgenic lines of N. benthamiana, which shows different expression level of GFP were tested to determine the threshold level for PTGS initiation. Agrobacterium-infiltration of GFP into those GFP-transgenic plants resulted in the co-silencing of the transgenic GFP. It was found that very low concentration of Agrobacterium with GFP and GFP+2b($OD_{600}$=0.002-0.02) which could not phenotypically induce an additive GFP expression, was enough to trigger PTGS pathway in all GFP transgenic plants. This strongly indicates that each GFP-transgenic plant should be expressing the transgenic GFP at its own pre-determined level and there was no buffer zone of additive GFP-expression to the threshold. In other words, the PTGS seems to be immediately activated as a self-defensive mechanism if an internal balance of gene expression is broken.

• Applied Biological Chemistry
• /
• 제37권5호
• /
• pp.343-348
• /
• 1994

A. tumafaciens C58로부터 $^3H-thymidine$으로 표지한 Ti plasmid를 분리하여 lyophilization-rehydration법으로 octadecyl rhodamine B로 표지한 liposome에 내포시켰다. Liposome조제에 사용한 지질은 phosphatidylserine(PS)을 사용한 PS liposome과, PS와 cholesterol(Chol)을 같은 mole비로 섞은 PS-Chol이었다. 꽃담배 원형질체는 0.1% macerozyme과 1.5% cellulase를 처리하여 유리시키고 불연속성 농도구배 원심분리로 정제하였다. $^3H-Ti plasmid$를 포함하고 있는 liposome$(1\;{\mu}mole\;PS)$을 5 mM $Ca^{2+}$과 10% PEG로 처리하여 꽃담배 원형질체$(10^6)$과 융합시켰다. 원형질체와 융합한 liposome의 양은 rhodamine B의 형광으로 측정하고 원형질체에 도입된 Ti plasmid의 양은 tritium의 방사능으로 측정하였다. 이때 PS liposome에서는 7.9%가 PS-Chol liposome에서는 7.2%의 liposome이 원형질체와 융합하였는데 융합과정에서 PS liposome서는 약 60%의 내용물이 유실되었고 PS-Chol liposome에서는 약 30%의 내용물이 유실되었다. 따라서 Ti plasmid를 식물원형질체에 도입하는데는 PS 보다는 PS-Chol로 구성된 liposome이 효율적인 것으로 나타났다. PS-Chol liposome을 사용할 경우에 1개의 원형질체에 약 1,700개의 Ti plasmid가 결합하고 있음으로 lyophilization-rehydration법을 사용하여 Ti plasmid를 식물원형질체에 도입할 경우에 식물의 형질전환 효율을 높일 수 있을 것임을 시사하여 준다.