Jin-Hyoung Lee

Molecular Characterization of Transgenic Rice Producing Resveratrol

Resveratrol, a plant phenolic compound, has potential therapeutic benefits due to its antioxidant properties. This is substantiated by previous studies that show that resveratrol derived from rice grains is an effective treatment agent for metabolic syndrome. Here, we characterized the T-DNA sequence, inserted T-DNA structure, copy number, integrity of the transgene locus, resveratrol synthase gene expression and resveratrol contents in the grains of two resveratrol transgenic rice lines, Iksan515 and Iksan526. The T-DNA transformation vector contained two expression cassettes of the resveratrol synthase gene under the control of the ubiquitin promoter and the bar selection marker gene under the control of the CaMV35S promoter. Flanking sequence analysis indicated that the T-DNAs were inserted into intergenic regions of chromosome 4 for Iksan515 and chromosome 12 for Iksan526. Two T-DNAs connected in an inverted repeat structure at a single locus of the rice genome were identified by whole genome sequencing and Southern blot hybridization in both Iksan515 and Iksan526. No novel open reading frames (ORFs) around insertion sites, sequences encoding allergenic or toxic protein, or other unintended effects by T-DNA insertion were found in either case. In addition, resveratrol synthase gene expression in leaves and resveratrol detection in brown rice grains suggested the successful expression of the inserted foreign resveratrol synthase gene in two transgenic rice lines.

Qualitative and quantitative PCR detection of insect-resistant genetically modified rice Agb0101 developed in korea

살충성 유전자 mcrylAcl을 포함하고 있는 해충저항성 유 전자변형 （GM) 벼 Agb0101이 국내에서 개발되었다． 향후 Agb0101 벼의 환경방출에 따른 모니터링과 이력추적을 위해서는 신뢰성 있는 검출방법의 개발이 필요하다． 따라서， 본 연구에서 해충저항성 GM벼의 사후 안전관리를 위한 정성적 및 정량적 PCR 검정 방법을 개발하였다． 벼 녹말분지효소 유전자 RBE4를 PCR 분석의 내재유전자로 사용하였고， 이의 primer쌍 RBEgh- 1/ - 2는 101bp의 PCR 증폭산물을 형성하였다． 정성 PCR 분석을 위해서 삽입된 T- DNA를 바탕으로 특이 Primer를 제작하였고， 이벤트 특이적 검출 primer의 경우 Agb0101의 도입유전자 및 벼 염색체 DNA 사이의 5``또는3``인접염기부위를 정확하게 특이적으로 PCR 증폭하였다． 반면， 대조구인 각종 작물， 국 내 벼 품종 및 Agb0101과 동일 형질전환 벡터를 갖는 해 충 저 항성 벼 에 서 는 어 떠 한 PCR 증폭산물도 형성하지 않았다. 표준물질로써 내재유전자 및 이벤트 특이적 단편으로 제조된 pRBECrR을 이용한 real- time PCR 분석에 의해 서 정량한계 （LOQ） 가 10 copies 농도의 범위인 것으로 확인되었고, 이의 유효성을 검증하기 위하여 상이한 농도 의 Agb0101시료 （1O, 5, 3 및 I%를 real-time PCR 분석하여 정량검정에 대한 표준편차 및 상대표준편차가 각각 0. 06~0.40 및 3.80, 7.01％ 의 낮은 범위에 포함되는 것을 확 인할 수 있었다． 이들 결과로 본 연구에서 개발된 정성 및 정량 PCR 검정 방법이 해충저항성 GM벼 Agb0101의 모니터링 및 이력추적에 효과적으로 이용될 수 있을 것으로 본다.

Development of transgenic rice lines expressing the human lactoferrin gene

Lactoferrin is an 80-kDa iron-binding glycoprotein that is found in high concentrations in human milk. Human lactoferrin (hLF) has several beneficial biological activities including immune system modulation and antimicrobial activity. In the present study, we devolved a method of hLF expression through introducing the hLF gene construct into Oriza sativa cv. Nakdong using the Agrobacterium-mediated transformation system. The expression of the hLF gene under the control of the rice glutelin promoter was detected in the seeds of transgenic rice plants. Transformed rice plants were selected on media containing herbicide(DL-phosphinothricin) and the integration of hLF cDNA was confirmed by Southern blot analysis. The expression of the full length hLF protein from the grains of transgenic rice plants was verified by Western blot analysis. The lactoferrin expression levels in the transformed rice grains determined by enzyme-linked immunosorbant assay accounted for approximately 1.5% of total soluble protein. Taken together, these data indicate that rice grains expressing hLF can be directly incorporated into infant formula and baby food.

T-DNA Inheritance Stability of Resveratrol Rice Iksan526 over Multi-generations

Confirmation of T-DNA inheritance and stability requires testing of several parameters such as phenotypic performance of transgenic lines, transgene integrity structure and expression stability for at least three consecutive generations, and Mendelian inheritance analysis, which are very important to transgenic breeding and commercialization of genetically modified crops (GMOs). In the present study, we measured nine agronomic and yield-related traits of Iksan526 and confirmed stable phenotypic performance comparing to the donor variety Dongjin over three years. Transgene integrity structure, flanking sequence, functional element copy numbers, absence of backbone sequence and transgene expression were all verified in Iksan526 over three consecutive generations, indicating a stable inheritance of the transgene. In addition, phenotypic and genotypic segregation were analyzed on F2 and F3 populations derived from a cross of Dongjin/Iksan526, and results complied with Mendel’s law of segregation based on single transgene locus. This study demonstrated detailed analyses and confirmation of transgene event Iksan526 which is crucial for commercialization of GMO.

Analysis of key nutrients and anti-nutrients in insect-resistant transgenic rice.

Content of key nutrients and anti-nutrients of the insect-resistant transgenic rice (Btt12R) developed in Korea that contains a cryIIIA insecticidal gene was compared with those of its non-transgenic counterpart (Oryza sative L. cv. Nakdongbyeo). Grains of Btt12R, its parent cultivar, and two commercial rice plants (cv. Yeonganbyeo and Hwaseongbyeo) grown in the adjoining fields under the same environmental conditions and field management were used for this study. Among the analyzed 47 nutrients (8 proximates, 17 amino acids, 8 fatty acids, 9 minerals, and 5 vitamins) and two anti-nutrients (trypsin inhibitors and phytic acid), although the levels of 17 components differed between Nakdongbyeo and Btt12R, all of the measured values from Btt12R were within the ranges of values observed in the two typical Korean varieties and commercial rice provided by the OECD. These results confirm that the nutritional quality of rice grains was not affected by the insertion of the cryIIIA gene.

Analysis of junction site between T-DNA and plant genome in Lissorhoptrus oryzophilus resistance GM rice

Abstract Four transgenic rice lines harboring insect-resistant gene cry3A showed ideal field performances characterized by high considerable resistance to rice water weevil ( Lis-sorhoptrus oryzophilus Kuschel). In this study, we estimated the insertion number of foreign genes, and analyzed the flanking sequences of T-DNAs in rice genome. As a result, T-DNA of BT12R1 line was inserted in exon region of rice chromosome 10. Two copies of T-DNAs were inserted in line BT12R2. BT12R3 line was analyzed at only left border flanking sequence. BT12R4 line was confirmed one copy of foreign gene insertion at the position 24,516,607 ~ 24,516,636 of rice chromosome 5, accompanied by a deletion of 30 bp known genomic sequences. This intergenic position was confirmed none of expressed gene and any deletion/addition of T-DNA sequence. In conclusion, these molecular data of rice water weevil resistant Bt rice would be used to conduct the biosafety and environment risk assessment for GM crop commercialization.

Transgene structures of marker-free transgenic Bt rice plants

Abstract A less simple approach developed for generation of marker-free transgenic plants is to select transformants without the use of selective marker genes. Some results about development of marker-free transgenic plants were obtained using a non-selective approach in several crops such as rice, potato and tobacco. However, the study did not provide evidence on detailed characterization of introduced gene on genome, a critical step for confirming the stable integration and transmission of a foreign gene. In this study, we evaluated structure and integration sites of transgene (m Cry1Ac ) in the transgenic Bt rice plants which were made via conventional Agrobacterium -mediated transformation by non-selective method. Structure and integration sites of transgene in these transgenic plants had similar fashion as those recovered under selection. Keywords Non-selective, Marker-free transgenic rice, Agrobacterium -mediated transformation, Transgene, integration site 서 론 선발마커유전자(Selective marker gene)는 목표유전자와 연결하여, 도입유전자가 형질전환 된 식물체를 선택적으로 선발하는데 사용된다. 통상적으로

Expression of Human Lactoferrin Gene in Transgenic Rice (Oryza sativa L.)

Lactoferrin is an 80-kDa iron-binding glycoprotein known to exert many biological activities, such as facilitating iron absorption and having antimicrobial and anti-inflammatory effects. Rice can be a useful target for edible food plants to introduce human lactoferrin, because it has lower allergenicity and is likely to be safer than microorganisms or transgenic animals. A cDNA fragment encoding human lactoferrin (HLF) driven by the maize polyubiquitin promoter, along with herbicide resistance gene (bar) driven by CaMV 35S promoter, was introduced into rice (Oryza sativa L. cv. Dong Jin) using the Agrobacterium -mediated transformation system. Putative transformants were initially selected on the medium containing bialaphos. The stable integration of the bar and HLF genes into transgenic rice plants was further confirmed through polymerase chain reaction (PCR) and Southern blot analyses. The expression of the full length HLF protein from various tissues such as grains and young leaves of transgenic rice was verified by Western blot analysis. Analysis of progeny also demonstrated that introduced genes were stably inherited to the next generation at the Mendelian fashion.