• Journal of Plant Biotechnology
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• 제37권3호
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• pp.275-282
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• 2010

고등식물의 엽록체 형질전환은 핵 형질전환에서 기대 할 수 없는 여러 가지 이점을 가진다. 외래 단백질의 발현율을 획기적으로 높일 수 있고, 여러 유전자를 동시에 발현시킬 수 있으며, 상동재조합에 의한 부위-특이적 유전자 삽입으로 인해 유전자 침묵 및 위치효과가 없다. 더욱이, 대부분 작물은 화분을 통한 도입된 유전자의 전이가 불가능한 모계 유전을 하기 때문에 엽록체 형질전환은 환경 친화적이다. 엽록체 형질전환 시스템은 핵 형질 전환과 달리 작물에서의 성공에 제한적이었으나 지난 10년 동안 이런 한계가 극복되어 콩, 당근, 상추 및 유채 등의 작물에서도 성공하게 되었다. 그러므로 이제 작물의 엽록체 형질전환은 농업적 형질의 개선뿐 만 아니라, 고부가가치 백신과 의료용 단백질 생산을 통한 의약품 산업의 성장에 활용될 수 있을 것이다.

• ALGAE
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• 제33권4호
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• pp.351-358
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• 2018

The ability to transform the chloroplast of Cyanidioschyzon merolae was limited by lack of confirmed and reliable promoter sequences (among other reasons), capable of delivering stable or modulated DNA transcription followed by protein synthesis. Our research has confirmed the applicability of three selected chloroplast promoters in C. merolae chloroplast overexpression of the exogenous protein (i.e., chloramphenicol acetyltransferase) and genetic transformation. These results might facilitate further research on genetically modified strains of C. merolae to envisage yet unknown aspect of cellular and plastic physiology as well as C. merolae potential applications as bio-factories or sources of useful chemicals.

• Journal of Plant Biotechnology
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• 제33권3호
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• pp.185-194
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• 2006

엽록체는 숙주세포에 잡아먹힌 (식균작용) 남세균이 숙주세포와 공생관계를 형성하여 온 것으로 간주된다. 엽록체 게놈은 정적이라고 이해하고 있지만 형질전환을 통하여 상동염기가 도입되면 이와는 반대로 intramolecular homologous recombination에 의해 subgenomic circle을 만드는 등 매우 다이나믹하다는 것이 최근에 증명되고 있다. 고등식물의 엽록체 형질전환은 핵 형질전환에서 기대할 수 없는 여러 이점을 제공한다. 예컨대, transgene의 발현율을 높일 수 있고, transgene들을 polycistronic하게 발현할 수 있으며, 도입된 transgene이 모계유전을 하게 된다는 것 등이다. 담배는 엽록체 형질전환의 모델 식물로 사용되어 왔으나 최근에는 벼, 대두, 면화 등 다른 주요 작물의 형질전환도 가능하게 되었다. 엽록체 형질전환된 작물은 미생물을 이용하여 고부가가치 단백질을 생산하는 생물반응기를 향후 대체할 수 있게 될 것이다.

• Journal of Plant Biotechnology
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• 제35권1호
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• pp.41-45
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• 2008

Laccase (EC 1.10.3.2) is a small group of enzymes that catalyze the oxidation of a broad range of phenolic compounds including hazardous and recalcitrant pollutants in the environment. This study attempted to develop an efficient system for production of a recombinant laccase by chloroplast genetic transformation of tobacco. Chloroplast transformation vector was constructed and introduced into the tobacco chloroplast genome using particle bombardment. Chloroplast-transformed plants were subsequently regenerated. PCR and southern blot analyses confirmed stable integration of the laccase gene into the chloroplast genome. Northern blot analysis revealed that mRNA of the laccase gene was highly expressed in chloroplast-transformed plants.

• Journal of Plant Biotechnology
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• 제46권4호
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• pp.323-330
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• 2019

The high expression level of industrial and metabolically important proteins in plants can be achieved by plastid transformation. The CaIA vector, a Capsicum-specific vector harboring aadA (spectinomycin resistance), is a selectable marker controlled by the PsbA promoter, and the terminator is flanked by the trnA and trnI regions of the inverted repeat (IR) region of the plastid. The CaIA vector can introduce foreign genes into the IR region of the plastid genome. The biolistic method was used for chloroplast transformation in Scoparia dulcis with leaf explants followed by antibiotic selection on regeneration medium. Transplastomes were successfully screened, and the transformation efficiency of 3 transgenic lines from 25 bombarded leaf explants was determined. Transplastomic lines were evaluated by PCR and Southern blotting for the confirmation of aadA insertion and its integration into the chloroplast genome. Seeds collected from transplastomes were analyzed on spectinomycin medium with wild types to determine genetic stability. The increased chloroplast transformation efficiency (3 transplastomic lines from 25 bombarded explants) would be useful for expressing therapeutically and industrially important genes in Scoparia dulcis L.

• Journal of Plant Biotechnology
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• 제33권4호
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• pp.233-236
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• 2006

Human serum albumin (HSA) is the most abundant protein in plasma and is the most often used intravenous protein in many human therapies. However, HSA is currently extracted only from plasma because commercially feasible recombinant expression systems are not available. This study attempted to develop an efficient system for recombinant HSA production by chloroplast transformation of tobacco. A HSA cDNA was isolated from a cDNA library constructed with human liver tissue. Chloroplast transformation vectors were constructed by introducing various regulatory elements to HSA regulatory sequences. Vectors were delivered by particle bombardment into leaf explants and chloroplast-transformed plants were subsequently regenerated into whole plants. Southern blot analysis confirmed that the HSA cDNA was incorporated between rps12 and orf70B of the chloroplast genome as designed. Western blot analysis revealed that hyper-expression and increasing the stability of HSA were achieved by modification of the regulatory sequences using the psbA5'UTRs in combination with elements of the 14 N-terminal amino acids of the GFP and the FLAG tag. However, only plants transformed with the vector containing all of these elements were able to accumulate HSA.

• Journal of Plant Biotechnology
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• 제34권3호
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• pp.271-275
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• 2007

엽록체 형질전환된 식물체를 얻으려면 먼저 세포수준에서 모든 엽록체가 형질전환되어야 하는데, 세포내에는 많은 수의 엽록체가 존재하므로, 엽록체 형질전환 벡터가 전이되어 형질전환된 엽록체는 선발배지에서 선택적으로 분열을 계속하고 형질전환되지 않은 엽록체들은 분열을 하지 못하게 되어, 결국 해당 세포내의 모든 엽록체가 형질전환된 상태에 이르게 된다. 따라서 만일 해당 세포내에 엽록체의 수가 적으면 그만큼 효율적으로 엽록체 형질전환을 할 수 있을 것이다. 본 연구에서는 담배의 FtsZ 유전자를 핵형질전환법으로 과잉 발현시킴으로써 엽록체의 분열이 저해되어 엽육세포내에 거대한 엽록체 3-5개를 가진 담배식물체의 엽육조직을 이용하여, 엽록체 형질전환을 한 결과, 엽록체 형질전환 빈도가 약 40% 증가되었다.

• Journal of Plant Biotechnology
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• 제38권1호
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• pp.42-48
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• 2011

고등식물의 엽록체 유전공학은 핵 형질전환과 비교해 볼때 여러 가지 독특한 장점을 가진다. 높은 transgene 발현율, 상동 재조합에 의한 site-specific transgene의 삽입으로 인해 유전자의 position effect가 없으며, 단일 형질전환으로 동시에 여러 유전자의 도입이 가능하고 모계 유전으로 인해 화분 방출 위험을 감소시킬 수 있다. 담배 specific한 pCtVG (trnI-Prrn-aadA-mgfp-TpsbA-trnA) 벡터를 이용하여 안정적인 감자 엽록체 형질전환 시스템을 개발하였다. 감자 엽록체 게놈으로 외래유전자의 삽입과 homoplasmic level은 PCR과 Southern blot 분석으로 확인하였다. Northern과 immunoblot 분석 및 GFP fluorescence imaging을 통하여 엽록체 형질전환체의 잎에서 GFP 유전자가 강하게 발현, 축적됨을 알 수 있었다. 본 연구에서 확립된 감자 엽록체 형질전환 시스템을 이용하여 유용 유전자를 도입함으로써 농업적 형질을 개선하거나 고부가가치 단백질을 대량 생산하는 감자를 보다 효율적으로 개발할 수 있을 것이다.

• Journal of Plant Biotechnology
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• 제3권3호
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• pp.113-121
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• 2001

The use of a marker gene in a transformation process aims to give a selective advantage to the transformed cells, allowing them to grow faster and better, and to kill the non-transformed cells. In general, the selective gene is introduced into plant genome along with the genes of interest. In some cases, the marker gene can be the gene of interest that will confer an agronomic characteristic, such as herbicide resistance. In this review we list and discuss the use of the most common selective marker genes on plant transformation and the effects of their respective selective agents. These genes could be divided in categories according their mode of action: genes that confer resistance to antibiotics and herbicides; and genes for positive selection. The contention of the marker gene flow through chloroplast transformation is further discussed. Moreover, strategies to recover marker-free transgenic plants, involving multi-auto-transformation (MAT), co-transformation, site specific recombination and intragenomic relocation of transgenes through transposable elements, are also reviewed.

• 한국식물학회:학술대회논문집
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• 한국식물학회 1999년도 제13회 식물생명공학심포지움 New Approaches to Understand Gene Function in Plants and Application to Plant Biotechnology
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• pp.79-84
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• 1999

Oxidative stress is one of the major environmental stresses to plants. Reactive oxygen species (ROS) generated during metabolic processes damage cellular functions and consequently lead to cell death. Fortunately plants have in vivo defense system by which the ROS is scavenged by enzymes such as superoxide dismutase (SOD) and ascorbate peroxidase (APX). In attempts to understand the protection mechanism of plant against oxidative stress, we developed transgenic tobacco (Nicotiana tabacum cv. Xanthi) plansts thet expressed both SOD and APX in chloroplast using Agrobacterum-mediated transformation and evaluated their protection capabilities against methyl viologen (MV, paraquat) -mediated oxidative damage. Three double transformants (CAI, CA2, and CA3) expressed the chimeric CuZnSOD and chimeric APX in chloroplast, and one transformant (AM) expressed the chimeric APX and chimeric MnSOD in chloroplast. In addition, we obtained three lines of transformants (C/Al, C/A2, and A/C) that expressed the APX and SOD than control plants, and more resistant to oxidative stress caused by MV. TRansformants (C/A and A/C) overexpressing MnSOD, CuZnSOD and APX at the same time showed the highest resistance to MV-mediated oxidative stress among the transformants.