Glutamine synthetase (GS) is an enzyme that plays an essential role in the metabolism of nitrogen by catalyzing the condensation of glutamate and ammonia to form glutamine. Exposure of plants to cadmium (Cd) has been reported to decrease GS activity in maize, pea, bean, and rice. To better understand the function of the GS gene under Cd stress in rice, we constructed a recombinant pART vector carrying the GS gene under the control of the CaMV 35S promoter and OCS terminator and transformed using Agrobacterium tumefaciens. We then investigated GS overexpressing rice lines at the physiological and molecular levels under Cd toxicity. The GS activity along with mRNA expression were found higher in transgenic than in wild type plants. And this is validated by the low malondialdehyde contents observed 10 days after treatment. GS overexpression in rice resulted in the modulation of expression of enzymes responsible for membrane peroxidation, which may result in the sudden death of plants. Our results thus describe the features of a transgenic rice plants with enhanced tolerance to Cd toxicity.

In this study, we generated and characterized the transgenic rice plant expressing a spider silk protein. A cDNA coding for the C-terminus of spider silk protein (AvMaSp) was cloned from the spider Araneus ventricosus. Analysis of the cDNA sequence shows that the C-terminus of AvMaSp consists of 165 amino acids of are petitive region and 99 amino acids of a C-terminalnon-repetitive region. The peptide motifs found in spider silk proteins, GGX and An, were conserved in the repetitive region of AvDrag. The AvMaSp cDNA was expressed as a 28kDa polypeptide in baculovirus-infected insect cells. To produce transgenic rice plant with high contents of glycine and alanine, the prolamin promoter-driven AvMaSp was introduced into rice plant via Agrobacteriumtumefaciens-mediated gene transformation. Because of seeds pecific prolamin promoter, expression of AvMaSp protein has been achieved inriceseed. The introduction and copy number of the AvMaSp gene in transgenic rice plants were determined by PCR and Southern blot analysis. AvMaSp expression in transgenic rice seeds was examined by Northern blot and Western blot analysis. Immuno fluorescence staining with the AvMaSpantiserum revealed that the recombinant AvMaSp proteins were localized in transgenic rice seeds. Furthermore, the amino acid content analysis showed that transgenic rice seeds were greatly increased in glycine and alanine as compared to controls. The present study is the first to show the expression of spider silk protein in rice seed.

Development of transgenic plant increasing crop yield or disease resistance is good way to solve the world food shortage. However, the persistence of marker genes in crops leads to serious public concerns about the safety of transgenic crops. In the present study, we developed marker-free transgenic rice inserted high molecular-weight glutenin subunit (HMW-GS) gene (Dx5) from the Korean wheat cultivar ‘Jokyeong’ using Agrobacterium-mediated co-transformation method. The Dx5’s own promoter was used for protein expression. Two expression cassettes comprised of separate DNA fragments containing only the Dx5 and hygromycin resistance (HPTII) genes were introduced separately into Agrobacterium tumefaciens EHA105 strain for co-infection. Each EHA105 strain harboring Dx5 or HPTII was infected into rice calli at a 3: 1 ratio of EHA105 with Dx5 gene and EHA105 with HPTII gene expressing cassette. Then, among 270 hygromycin-resistant transformants, we obtained 27 transgenic lines inserted with both the Dx5 and HPTII genes into the rice genome. We reconfirmed integration of the Dx5 gene into the rice genome by Southern blot analysis. Wheat Dx5 transcripts in T1 rice seeds were examined with semi-quantitative RT-PCR. Protein expression of the Dx5 was analyzed with Western blot using polyclonal antibody recognising x-type of glutenin subunits in T1 seeds. It was suggested that the protein-processing system was conserved between rice and wheat. Finally, the marker-free plants containing only the Dx5 gene were successfully screened at the T1 generation.

UDP-glucose 4-epimerase catalyzes the reversible conversion of UDP-glucose to UDP-galactose. The gene, named BrUGE1, isolated from a Chinese cabbage composes of a total length of 1,328 bp that contains a single open reading frame (ORF) of 1,056 bp which encodes a polypeptide of 351 amino acid residues with a calculated mass of 39.0 kDa. Expression analysis showed that BrUGE1 is tissue specific and highly expressed in stem of rice plant. Interestingly, BrUGE1 mRNA was highly accumulated by drought stress with significantly higher amount of soluble sugar. Morphological evaluation showed an increase in yield and yield components compared to the wild type. Moreover, a better growth performance on galactose as well as higher UGE1 expression was observed in transgenic rice lines than in wild type. In the Ubi-1::BrUGE1 lines, the increase of UGE1 expression was apparently sufficient to overcome the toxic effects of galactose. Taken together, the Ubi-1::BrGUE1 rice lines increased yield probably by increasing the rate of filled grains. The enhanced drought tolerance may be due to the induction of soluble sugar which may act as osmolyte to compensate dehydration during drought stress.

Bacterial blight is a serious problem of rice in irrigated and rainfed lowlands. It is caused by Xanthomonas oryzae pv. oryzae (Xoo) which is represented by many pathotypes, making it difficult to control. Plant proteases are important players in immunity acting either in the execution of attack, in signaling cascade or in perception of invader. This study demonstrates the response of cysteine protease (CP) upon interaction with the pathogen. The cysteine protease encoding full-length cDNA was identified and characterized using web-based tools. Conserved domain of the gene revealed its affinity to Peptidase_CIA family. The full-length cDNA of CP in Brassica rapa was then cloned and overexpressed in rice. Insertion of gene was verified in the transformants through PCR assay. Spatiotemporal expression of the gene was performed in transgenic rice. To evaluate the resistance of CP-overexpression lines to Xoo, transgenic plants were inoculated with two races of Xoo. In planta analysis of enzymatic activity of CP was also performed before and after infection by the pathogen.

국내 대학과 국립식량과학원 간의 협력 연구를 통해 확보된 32개 벼 형질전환 계통들 중 작물학적 성능이 유망한 11개 계통들에 대해 작물학적 특성과 미질 특성이 평가되었다. 주요 결과는 아래와 같다.1. 이들 계통들은 수량성, 환경스트레스저항성, 내병성에관여하는 7종의 유전자가 니폰바레, 낙동벼, 동진벼등에 형질전환 되어 육성된 계통들이다.2. 미질 특성과 관련된 11개 조사형질을 이용한 다변량분석결과, 형질전환에 이용된 모품종과 도입유전자들에 대한 각 형질전환 계통군들의 뚜렷한 집구현상은관찰되지 않았다.3. 실용적 측면에서는 작물학적 특성이 모품종과 유사하면서 미질 특성이 양호한 계통이 다수 확인되었다.4. 그러나 모품종의 유용한 작물학적 특성, 즉 ‘상품성’을온전히 견지하고 있다고 판단되는 계통은 선정하기 어려웠다.5. 도입할 유전자의 선정과 유전적으로 고정된 형질전환계통을 확보하는데 소요되는 경비와 시간을 고려할때, 향후 우량 벼 형질전환 계통의 육성효율을 제고하기 위해서는 기존에 비해 보다 구체적이며 차별화 된육종전략의 개발이 필요하다고 판단되었다.

국내 대학과 국립식량과학원 간의 협력 연구를 통해 확보된 32개 벼 형질전환 계통들에 대하여 주요 작물학적 특성및 수량성을 평가하였다. 이들 계통들은 수량성, 내병성, 스트레스저항성, 미질변이 등에 관여하는 17종의 유전자가 니폰바레, 낙동벼, 동진벼 등에 형질전환 되어 육성된 계통들이다. 주요 결과는 아래와 같다.1. 형질전환 계통들은 출수기, 간장, 수장, 수수 등의 주요작물학적 특성뿐만 아니라 수량성에서도 모품종과 상당한 차이를 보였다.2. 조사된 벼 형질 전환계통들에서 조사된 농업형질에 대한다변량분석 결과, 모품종에 비해 각 형질전환 계통군 내에서 관찰된 작물학적 상이성은 일정한 방향성이 없는무작위적이었으며, 그 변화폭도 3개의 모품종들의 작물학적 차이보다 훨씬 크게 관찰되었다.3. 따라서 우량 벼 형질전환 계통 육성의 효율성을 제고하기 위해서는 모품종에 형질전환 되어 발현될 유전자의우수성뿐만 아니라, 계통육성과정에서 다양한 유전학적요인에 의해 야기되는 ‘표현형 왜곡현상’을 적절히 제어할 수 있는 육종전략도 함께 고려되어야 할 것으로 사료되었다.

가뭄저항성벼의 복수세대에 대한 후대안정성을 서던 블롯과 PCR로 분석한 결과, 가뭄저항성벼의 CaMsrB2-8 T4 ~ T6 세대에서는 도입된 모든 유전자들이 안정적으로 도입되어 있으며, T-DNA 구성요소 이외의 backbone DNA는 가뭄저항성벼에 삽입되지 않았음을 확인하였다. 목적 유전자인 CaMsrB2와 제초제 저항성 선발 마커인 bar가 가뭄저항성벼의 CaMsrB2-8 T4 ~ T6 세대에서 안정적으로 발현됨을 검증하였다. 제초제 저항성 선발 마커로 도입된 PAT 단백질의 발현 분석 결과에서도 CaMsrB2-8 T4 ~ T6의 3세대에서 생육시기별, 부위별로 안정적으로 발현됨을 입증하였다. 도입유전자의 삽입 위치를 확인한 결과, 가뭄저항성벼 CaMsrB2-8의 도입유전자가 벼 1번 염색체 내에서 intergenic한 상태로 안정적으로 유지되고있음을 확인하였다. 이상의 분석 기법을 통해 복수세대에서 가뭄저항성벼의 도입 유전자들이 안정적으로 유지되고 목적 단백질들이 안정적으로 발현되고 있음을 확인하였다.

본 연구는 벼에서 양성자 펌프의 활성을 증가시키는 것으로 알려진 오옥실 결합단백질 ABP57 유전자의 과발현에 따른 분자생물학적 특성을 분석해 작물 분자육종을 위한 기초자료로 활용하고자 수행하였다.ABP57 유전자 과발현 형질전환체와 대비 품종 낙동벼의 뿌리조직에서 유전자 발현 변화를 비교 분석한 결과는 다음과 같다. 두 식물체간에 발현량이 2배 이상 차이를 보인 유전자 수는 29,389개 중 총 148개였으며, 그 가운데 65개는 발현량 증가를 보였다. ABP57 유전자 과발현 형질전환체에서 발현량 변화를 보인 유전자들의 생물적 기능을 분석하기 위해 유전자 온톨로지 분석을 수행한 결과 각 부문별로 생물적 작용 부문에 67개 세부항, 세포 구성요소 부문에 59개 세부항, 분자기능 부문에 29개 세부항에 각각 영향을 미치는 것으로 나타났다. ABP57 유전자 과발현에 따라 세 개 부문 모두에서 공통적으로 나타난 영향은 H+-ATPases의 활성 증가, 여기에 필요한 에너지원 관련 화합물(ATPs, purines) 생합성 및 대사관련 활동 증가, 이온의 막투과 및 운반체 활동 증가 등이 확인되어 Kim et al.(2001)의 선행 연구결과와 일치하는 경향이었다. 그러나, 엽록체의 일부 구성요소나 G-protein 결합 단백질 신호전달경로, cytokine 신호전달 경로 등과 관련된 단백질 활성 감소는 식물 생육에 부정적 영향을 미칠 것으로 추정된다.따라서, 향후 작물의 효율적 분자육종을 위해서는 개별 유전자의 명확한 기능 분석과 더불어 유전자 상호작용에 대한 네트워크 분석 등에 대한 연구개발 노력과 함께 형질전환 식물체에 대한 생리적 특성, 표현형, 농업형질 특성 등에 대한 효과적 분석을 위해 전통육종과의 지속적 교류와 협력이 반드시 필요할 것이다.

Farmers have use phosphate fertilizer to provide sufficient yields. However, overuse of phosphorus accumulate in soil and causes soil and water pollution. We evaluated the phosphate acquisition and growth characteristics of OsPT1 transgenic rice (OsPT1-OX, over-expressing the high affinity phosphate transporter 1) in high phosphate soils with different level of nitrogen fertilizer treatment to investigate removing ability of excessive phosphate from soil. OsPT1-OX had shorter culm length but more tillers than those of wild-type plants in each soil conditions. Phosphate content per dry weight of OsPT1-OX was 1.8 times higher than that of wild-type under control fertilizer treated conditions. Although the dry weight of OsPT1-OX was not different from that of wild-type plants, whole plant phosphate content was 1.7 times higher than that of wild-type plants under control fertilizer conditions. Tiller number and phosphate content per dry weight of wild-type plants increased following high levels of phosphate application but did not change by following additional nitrogen application. Tiller number and phosphate content per dry weight of OsPT1-OX did not change under the high phosphate condition, but increased following nitrogen application under similar conditions. Whole plant phosphate content was highest under high nitrogen and high phosphate application conditions. These results suggest that OsPT1-OX may reduce phosphate content in soils containing excess phosphate and may be further effective under high nitrogen condition.

Rice is one of the most important major food crops which provide the major food for more than half of global population. To improve the grain quality as well as grain yield has been the essential breeding goal in rice. The composition of amylopectin is the determinant of rice eating quality under certain threshold of protein content and the ratio of amylose and amylopectin. In this study, RBE 1 driven by CaMV-35S promoter was constructed and transformed using Agrobacterium tumefaciens. We selected single copy with low amylose content among transgenic lines. The mRNA expression was investigated using RT-PCR, and enzyme activity was determined using activity staining method in mid-milky stage endosperm. Also, the overexpression vectors for RBE 1 and SSS 1 driven by seed specific globulin promoter were constructed, respectively. Moreover, the RNA interference vectors for soluble starch synthase 1 and granule bound starch synthase 1 derived by CaMV35S promoter were constructed, respectively and transformed using Agrobacterium tumefaciens. The transgene has been confirmed by amplification of HPT and target gene. The transgenic plants obtained will be used to investigate the gene function of related starch pathway in plant cells using Gopumbyeo as a wild type rice, based on the gain-of-function and the loss-of-function. The development of designed site-specific endonucleases boosted the establishment of gene targeting (GT) techniques in a row of different species. However, the methods described in plants require a highly efficient transformation and regeneration procedure and, therefore, can be applied to very few species. Here, we describe a highly efficient GT system that is suitable for all transformable plants regardless of transformation efficiency. Efficient in planta GT was achieved in rice by expression of a site specific endonuclease (SSS1::ZFN) that not only cuts within the target but also the chromosomal transgenic donor, leading to an excised targeting vector.

In this study, we generated and characterized the transgenic rice plant expressing a spider silk protein. A cDNA coding for the C-terminus of spider dragline silk protein (AvDrag) was cloned from the spider Araneus ventricosus. Analysis of the cDNA sequence shows that the C-terminus of AvDrag consists of 165 amino acids of are petitive region and 99 amino acids of a C-terminalnon-repetitive region. The peptide motifs found in spider drag line silk proteins, GGX and An, were conserved in the repetitive region of AvDrag. The AvDrag cDNA was expressed as a 28kDa polypeptide in baculovirus-infected insect cells. To produce transgenic rice plant with high contents of glycine and alanine, the prolamin promoter-driven AvDrag was introduced into rice plant via Agrobacteriumtumefaciens-mediated gene transformation. Because of seeds pecific prolamin promoter, expression of AvDrag protein has been achieved inriceseed. The introduction and copy number of the AvDrag gene in transgenic rice plants were determined by PCR and Southern blot analysis. AvDrag expression in transgenic rice seeds was examined by Northern blot and Western blot analysis. Immuno fluorescence staining with the AvDrag antiserum revealed that the recombinant AvDrag proteins were localized in transgenic rice seeds. Furthermore, the amino acid content analysis showed that transgenic rice seeds were greatly increased in glycine and alanine as compared to controls. The present study is the first to show the expression of spider silk protein in rice seed.

The significance of genetic stability and bio-safety environment has been recently recognized by many GM plants. This study was to evaluate the GM stability of transgenic rice and to identify the environment variance. The GM rice of vitamin A　-enriched rice and four check cultivars were analyzed the data on agronomic characters and principal component for 2009-2011 in large-GM crop field. Cultivation environment was conducted in the large-GM field and greenhouse to determine grain characters. In this experiment, there was no significant difference in agronomic characters between GM rice of vitamin A-enriched rice and a donor plant, Nagdong. Related to grain characters, grain appearance and physicochemical characteristics were similar to GM rice of vitamin A-enriched rice and a donor plant, Nagdong. However, grain appearance in GM rice of vitamin A-enriched rice showed to white core and white belly when GM rice of vitamin A-enriched rice was planted in greenhouse. The type and distribution of dominant weed species also were not different from GM rice of vitamin A-enriched rice and a donor plant, Nagdong. Additionally that of gene flow was not detected in dominant weed species by PCR analysis.

Regulations in the EU, Japan, Korea, etc. require that foods and feeds made of or derived from genetically modified organisms (GMOs) should be approved and labeled according to a threshold. Recently, disease resistant transgenic rice was developed in Korea, which resulted from the transformation events involving choline kinase gene, OsCK1. In order to monitor unintended release of the developed GM rice in the near future, as well as to meet GM-labeling requirements, the development of a reliable method for detection of disease resistant GM rice is requisite. Here, specific primer pairs for the detection of GMO was designed on the basis of a introduced gene and the flanking junction sequences between a plant DNA and a integrated gene construct, and also SPS gene was used as an endogenous reference material. Specificities of all designed primers were tested through qualitative PCRs. Clearly, target specific amplicons could be detected from disease resistant GM rice event. In addition, the limits of detection (LOD) using the event-specific primers were approximately 0.1% for the disease resistant GM rice line. This result indicated that the developed detection method is suitable for the traceability of disease resistant GM rice, because of using the primer specifically corresponded to the junction site between plant genomic DNA and inserted DNA. Keywords: genetically modified organisms, disease resistant GM rice, PCR detection, event-specific primer

The latest report on draft genome of Brassica rapa sequence has been published. To elucidate the functions of a large population of these genes and to search efficiently for agriculturally useful genes, the Full-length cDNA Over-eXpressor (FOX) gene hunting system was used. The FOX library was transformed into rice by Agrobacteriummediated transformation. Approximately 1,150 FOX-rice lines were generated. Genomic PCR analysis indicated that the average length of FL-cDNAs was 900∼1,200 bp with functional annotation of many unknown function (35.5%). Most of the randomly selected transgenic rice lines showed overexpression (92%) and barely mRNA expression in the wild type Gopum. Moreover, 94% of the 850 transgenic rice lines were moderately tolerant (slightly yellow) to cold and 9 lines were tolerant (seedling light green). For the salinity evaluation, most of the transgenic lines (85%) were highly susceptible whereas seven lines were tolerant. In addition, morphological evaluation of rice lines showed minimal phenotypic alteration (12%). About 25.1 and 22% were earlier in terms of days to heading and chlorophyll contents, respectively. Further, 18% of FOX rice lines showed lower chlorophyll contents. Filled grains, number of tillers, panicle length, culm and plant height were relatively less variable among the lines. These results provided useful genes for functional analyses in the mechanisms of identified transgenic tolerant lines.

The antimicrobial peptide possesses defence system to virus, fungi and bacteria. To study antibiotic in plant, antimicrobial peptides were obtained by PCR analysis by primers designed from antimicrobial peptides (Gene bank accession no. NM-004345), cloned in pET28 expression vector and the vector transformed into E. coli. And this gene was inserted into Ti-plasmid VB2 vector, which contained the pGD1 promoter. The expression construction was transformed into Agrobacterium EHA105 and then plant tissues of rice (Oryza sativa). Seeds from transgenic plants (T0) were germinated on selective media containing spectinomycin 50 mg/L. Selected plants and wild type were analyzed by PCR and RT-PCR with pGD1 promoter region and transgene specific primer set. All transgenic plants showed expression pattern of similar levels. We showed that the chromobody is effective in binding GFPand antimicrobial peptide gene in tobacco leaf. Most interestingly, this can be applied to interfere with the function of GFP fusion protein and to mislocalize (trap) GFP fusions to the plant cytoplasm in order to alter the phenotype mediated by the targeted proteins. Bacterial blight disease was enhanced resistance in transgenic lines. These results showed that antibiotic peptides might show a broad-spectrum antimicrobial activity.

The rice water weevil (RWW), Lissorhoptrus oryzophilus are major pests of aquatic rice plant in Korea as well as throughout the country. Larvae of RWW sucking the nourishment on roots, causes a stunted root system and reduces grain yields. To prevent these damages, we constructed various plant expression vectors, which were harbored by insecticidal genes, cryBP1 and cryIIIa, and fused with the actin promoter and/or the modified RCg2 root-preferential promoters for expressing the insect-toxic genes in leaves and roots. A cryBP1 was cloned from Bacillus popilliae, producing crystal toxin against Japanese beetle, and CryIIIa was modified from the δ-endotoxin gene of Bacillus thuringiensis ssp. tenebrionis, encoding the coleoptera-specific toxin. The vectors containing the insecticidal genes were transferred into Oryza sativa japonica cultivar, Nakdong, by Agrobacterium -mediated transformation method. Several independent transgenic lines were selected by Southern blotting and Western blotting, confirming that cryBP1 and cryIIIa genes were stably integrated into the plant genomes and were expressed in transgenic plants. Upon insect bioassay using RWW, the mortality of insect larvae on cryBP1 and cryIIIa transgenic rice lines recorded up to 41% and 34%, respectively. These results suggested that the transgenic lines can be used to develop Coleoptera-resistant cultivars and could be valuable for later application in crop breeding for insect resistance.

To characterize CBF/DREB1-homologue in rice, nine OsDREB1 genes have been identified and characterized in this lab. Among these, it was shown that OsDREB1D was induced by drought and slightly by cold stress. We found that OsDREB1A, -1D, and -1E could up-regulate OsDhn1:LUC construct in transactivation assay using rice protoplasts. Transgenic rice plants overexpressing OsDREB1D under the maize ubiquitin promoter (Ubi:OsDREB1D) revealed an enhanced stress tolerance to drought. We also generated transgenic rice of OsDREB1D under OsPOX1 promoter (OsPOX1:OsDREB1D), which is cold stress inducible preferentially in the reproductive organs of rice. We are currently examining the mechanism of the enhanced tolerance of the transgenic plants to drought stress using both molecular physiological and biochemical techniques.