• 한국작물학회:학술대회논문집
• /
• 한국작물학회 2022년도 추계학술대회
• /
• pp.194-194
• /
• 2022

Recently, food-induced allergies have emerged as major global concerns. In the past ten years, it has doubled in western nations, and it has also increased in Asia and Africa. In many cases of food allergy, peanut allergy is prevalent, typically permanent, and frequently life-threatening. Therefore, we utilized gene editing techniques on the three major allergen genes in peanuts, Ara h 1, Ara h 2, and Ara h 3. Using gibson assembly and golden gate assembly, we created two vectors, the gRNA-tRNA array CRISPR-Cas9 system and Prime-editing. Using LBA4404 strain and agrobacterium-mediated transformation, the vectors were transferred to two elite Korean peanut lines. After co-cultivation and tissue culture, we extracted the tissue cultured peanut DNA amplified the hygromycin resistance gene and Cas9 gene in the T-DNA region. The integration of the T-DNA region into the host genome was demonstrated by the presence of a specific band in some samples. There have only been a few reported peanut gene editing studies. So, this study will contribute to peanut allergy and gene editing research.

• 한국작물학회:학술대회논문집
• /
• 한국작물학회 2022년도 추계학술대회
• /
• pp.200-200
• /
• 2022

Recently, food-induced allergies have emerged as major global concerns. In the past ten years, it has doubled in western nations, and it has also increased in Asia and Africa. In many cases of food allergy, peanut allergy is prevalent, typically permanent, and frequently life-threatening. Therefore, we utilized gene editing techniques on the three major allergen genes in peanuts, Ara h 1, Ara h 2, and Ara h 3. Using gibson assembly and golden gate assembly, we created two vectors, the gRNA-tRNA array CRISPR-Cas9 system and Prime-editing. Using LBA4404 strain and agrobacterium-mediated transformation, the vectors were transferred to two elite Korean peanut lines. After co-cultivation and tissue culture, we extracted the tissue cultured peanut DNA amplified the hygromycin resistance gene and Cas9 gene in the T-DNA region. The integration of the T-DNA region into the host genome was demonstrated by the presence of a specific band in some samples. There have only been a few reported peanut gene editing studies. So, this study will contribute to peanut allergy and gene editing research.

• 한국해양바이오학회지
• /
• 제16권1호
• /
• pp.55-62
• /
• 2024

Various antimicrobial peptides (AMPs) from microalgae have shown antibacterial, antiviral, antifungal, anticancer, and antioxidant effects, and play crucial roles in medical applications, aquaculture-related disease management, and the food industry. Magainin 2 (MAG2), an AMP, exhibits high antibacterial and antitumor activity, necessitating an efficient recombinant expression system for low-cost, large-scale production. To enhance MAG2 secretion efficiency in Chlorella, we constructed the SS:MAG2:His vector using the known Chlamydomonas reinhardtii CA1 signal sequence (SS) and obtained a stable transformant via an Agrobacterium-mediated transformation method and RT-qPCR. ELISA results revealed that the MAG2 content secreted into the medium by the SS:MAG2:His transformants increased proportionally with mRNA expression. These findings offer a strategy for high MAG2 secretion in the Chlorella vulgaris platform, potentially minimizing downstream processing costs.

• 한국식물학회:학술대회논문집
• /
• 한국식물학회 1985년도 워크샵 및 심포지엄 북한산국립공원의 식생
• /
• pp.59-71
• /
• 1985

In rice, limited efforts have been made to identify genes by the use of insertional mutagens, especially heterologous transposons such as the maize Ac/Ds. We constructed Ac and gene trap Ds vectors and introduced them into the rice genome by Agrobacterium-mediated transformation. In this report, rice plants that contained single and simple insertions of T-DNA were analyzed in order to evaluate the gene-tagging efficiency. The 3'end of Ds was examined for putative splicing donor sites. As observed in maize, three splice donor sites were identified at the 3'end of the Ds in rice. Nearly 80% of Ds elements wered excised from the original T-DNA sites, when Ac cDNA was expressed under a CaMV 35S promoter. Repetitive ratoon culturing was performed to induce new transpositions of Ds in new plants derived from cuttings. About 30% of the plants carried at least one Ds that underwent secondary transposition in the later cultures. 8% of transposed Ds elements expressed GUS in various tissues of rice panicles. With cloned DNA adjacent to Ds, the genomic complexities of the insertion sites were examined by Southern hybridization. Half of the Ds insertion sites showed simple hybriodization patterns which could be easily utilized to locate the Ds. Our data demonstrate that the Ac/Ds mediated gene trap system could prove an excellent tool for the analysis of functions of genes in rice. We discuss genetic strategies that could be employed in a largee scale mutagenesis using a heterologous Ac/Ds family in rice.

• The Plant Pathology Journal
• /
• 제20권3호
• /
• pp.206-211
• /
• 2004

Transgenic Nicotiana benthamiana plants harboring coat protein (CP) gene of Zucchini green mottle mosaic virus (ZGMMV) were generated for virus-resistant screening and complementation analysis of related viruses for environmental safety assessment (SA) of living modified organism (LMO) purposes. Transformation of leaf disc of N.benthamiana was performed by using Agrobacterium-mediated method and the pZGC-PPGA748 containing the ZGMMV CP and NPTII genes. Two kinds of transgenic homozygous groups, virus-resistant and virus-susceptible N.benthamiana lines, were obtained by screening of challenging homologous virus for Tl generations. These two pathologically different lines can be useful for host-virus interactions and LMO environmental SA.

• 화훼연구
• /
• 제18권1호
• /
• pp.1-8
• /
• 2010

호접란의 형질전환시스템을 확립하기 위한 제 연구를 수행하였다. 항생제 kanamycin, hygromycin 및 spectinomycin 농도(0, 25, 50, 100, 200, and $400mg{\cdot}L^{-1}$)가 품종별 PLB 생존율에 미치는 영향을 알아보기 위한 실험에서 hygromycin은 $25mg{\cdot}L^{-1}$에서 모든 품종이 괴사하였으므로 형질전환 개체의 선발 항생제로는 hygromycin이 유리할 것으로 보였다. P. 'Maki Watanabe'와 P. 'Brother Lawrence' 두 품종에서 형질전환체 선발을 위한 DL-Phosphinothricin (PPT)의 적정 농도는 $0.5mg{\cdot}L^{-1}$이었다. 형질전환시 가장 높은 효율을 얻기 위한 공동배양 일수를 결정하기 위한 실험은 Dtps. 'City Girl'과 A. tumefaciens LBA4404를 이용하여 2단계로 이루어졌다. 균주와 VW 배지의 1 : 10 현탁액에 균주와 PLB를 감염시킨 결과 1시간 처리구에서 PLB 생존이 가장 많았다. 그런 다음 공동배양한 결과 5일 배양에서 PLB 생존수가 가장 많았지만, 4일 이상의 공동배양할 경우 PLB 조직이 연화가 되고 약해져서 죽게 되었다. 따라서 오히려 3일 공동배양 기간이 적당한 것으로 판단되었다. 박테리아 균주의 종류가 호접란 PLB의 형질전환에 미치는 효율을 비교하기 위해 A. tumefaciens LBA4404(pTOK233)와 EHA105(pGA643)를 이용하였다. LBA4404 보다 EHA105로 감염시킨 PLB의 생존율이 더 높았다. A. tumefaciens LBA4404(pTOK233)와 AGL1(pCAMBIA3301)을 이용한 형질전환 실험에서 치상된 PLB가 초기에 백변하는 정도가 LBA4404를 이용한 경우 눈에 띄게 빠르게 나타났고 새로운 PLB가 유도되는 정도도 매우 낮았다(1% 미만). 반면에 AGL1을 이용한 경우 40% 정도의 새로운 PLB 및 유식물체 형성율을 나타내었다. 형질전환 실험에서 최종적으로 hygromycin 저항성 식물체 11개체와 PPT 저항성 식물체 32개체를 얻어냈으나 진정한 형질전환체인지는 차후에 더 검정이 되어야 할 것으로 보인다.

• 한국식물병리학회:학술대회논문집
• /
• 한국식물병리학회 2003년도 정기총회 및 추계학술발표회
• /
• pp.148.2-149
• /
• 2003

Cucumber fruit mottle mosaic tobamovirus (CFMMV) causes severe mosaic symptoms with yellow mottling on leaves and fruits, and occasionally severe wilting of cucumber plants. No genetic source of resistance against this virus has been identified. The genes coding for the coat protein or the putative 54-kDa replicase were cloned into binary vectors under control of the SVBV promoter. Agrobacterium-mediated transformation was peformed on cotyledon explants of a parthenocarpic cucumber cultivar with superior competence for transformation. R1 seedlings were evaluated for resistance to CFMMV infection by lack of symptom expression, back inoculation on an alternative host and ELISA. From a total of 14 replicase-containing R1 lines, 8 exhibited immunity, while only 3 resistant lines were found among a total of 9 CP-containing lines. Line 144 homozygous for the 54-kDa replicase was selected for further resistance analysis. Line 144 was immune to CFMMV infection by mechanical and graft inoculation, or by root infection following planting in CFMMV-contaminated soil. Additionally, line 144 showed delay of symptom appearance following infection by other cucurbit-infecting tobamoviruses. Infection of line 144 plants with various potyviruses and cucumber mosaic cucumovirus did not break the resistance to CFMMV. The mechanism of resistance of line 144 appears to be RNA-mediated, however the means is apparently different from the gene silencing phenomenon. Homozygote line 144 cucumber as rootstock demonstrated for the first time protection of a non-transformed scion from soil inoculation with a soil borne pathogen, CFMMV.

• Plant Biotechnology Reports
• /
• 제1권1호
• /
• pp.19-25
• /
• 2007

To develop an efficient screening method for detection of the transgene in Chinese cabbage (Brassica rapa spp. pekinensis) utilizing Basta spray, optimal conditions for Basta application were examined in this study. Two transgenic Chinese cabbage lines were obtained through Agrobacterium-mediated transformation and used as transgenic positive controls in the Basta screening experiment. Differential concentrations of glufosinate-ammonium were sprayed into three different growth stages of 12 commercial Chinese cabbage cultivars. The results showed that no plants could survive higher than 0.05% glufosinate-ammonium, and plants at the 2-3 leaf stage were most vulnerable to glufosinate-ammonium. On the other hand, no damage was observed in the transgenic control plants. Reliability of the Basta spray method was proven by showing perfect co-segregation of the tolerance to glufosinate-ammonium and the presence of the bar gene in T₁ segregating populations of the transgenic lines, as revealed by both PCR and Southern blot analyses. Using the developed Basta screening method, we tried to investigate the transgene flow through pollen dispersal, but failed to detect any transgene-containing non-transgenic Chinese cabbages whose parents had been planted adjacent to transgenic Chinese cabbages in field conditions. However, the transgene was successfully detected using Basta spray from the non-transgenic plants bearing the transgene introduced by hand-pollination. Since the Basta spray method developed in this study is easy to apply and economical, it will be a valuable tool for understanding the mechanism of gene flow through pollen transfer and for establishing a biosafety test protocol for genetically modified (GM) Chinese cabbage cultivars.

• 한국육종학회지
• /
• 제49권3호
• /
• pp.170-179
• /
• 2017

분자농업은 식물을 일종의 공장개념으로 확대 적용하여 산업적으로 가치가 높은 유용물질들을 대량생산하는 분야로 최근 많은 각광을 받고 있다. 그 중에서 벼 캘러스를 이용한 단백질 발현 시스템은 대량 배양이 가능하고 목적 단백질의 높은 발현율로 인한 산업화가 가능한 기술이다. 본 연구는 이러한 벼(Oryza sativa L.) 캘러스의 활용도를 높이기 위한 효율적인 형질전환 시스템을 구축하기 위해 수행되었다. 형질전환에 이용된 종자의 종피 제거 시 손을 이용함으로써 캘러스 유도율을 높여주었고, 6년 정도의 오래된 종자에서도 원활한 캘러스 유도가 가능하였다. 목적 유전자가 도입된 캘러스의 선발은 최소한 3주 이상의 배양기간을 필요로 하였고 가장 효율적인 것은 한번의 계대를 포함한 6주 배양 후 선발하는 것이었다. 이러한 선발은 유전자가 식물세포의 genomic DNA 안에 안정적으로 삽입되어 이루어졌음을 서던 블롯 분석 및 후대검정 등을 통하여 확인할 수 있었다. 이러한 장기적이고 대량의 배양이 가능한 벼 캘러스의 효율적인 선발 시스템은 벼를 이용한 유용 재조합 단백질의 산업화에 실속있는 기술로 기여할 수 있을 것이다.

• Journal of Plant Biotechnology
• /
• 제40권3호
• /
• pp.135-140
• /
• 2013

마커프리 형질전환 식물체 개발 방법의 하나인 무선발 형질전환은 선발마커 유전자를 이용한 도입유전자의 선발과정 없이, 연쇄중합반응(PCR)과 같은 직접 분석방법을 사용해 도입유전자가 도입된 형질전환 식물체를 개발하는 방법이다. 현재까지 감자, 담배, 알팔파, 밀, 땅콩 등 여러 작물에서 무선발 형질전환을 이용한 마커프리 형질전환체가 보고되었다. 그러나 대부분 무선발 형질전환체의 분석이 $T_0/T_1$세대에서의 PCR을 이용한 도입유전자 확인과 후대검정으로 수행되어, 유전자의 안정적 도입 확인을 위한 분자생물학적 특성평가 결과는 보고된 바 없다. 본 연구에서는 4계통의 무선발 형질전환 Bt벼에 대한 도입유전자 특성을 분자생물학적 방법으로 검정하여 유전자도입 위치, 도입유전자의 구조, 벡터 삽입여부 등의 분석 결과를 제시하였다.