형질전환 벼와 일반 벼의 환경위해성 평가 기초자료를 위해 경상남도 사천시에서 2013~2017년, 경상북도 군위군에서 2015~2016년 7월 말부터 10월 초까지 벼에 발생하는 곤충상을 조사하고 군집특성을 분석하여 비교하였다. 사천시에서 형질전환 벼는 경상대학교 LMO (Living genetically Modified Organism) 격리포장 내에서 재배되었고, 일반 벼는 경상남도 사천시 사천읍 두량리에서 재배되었다. 군위군에서 형질전환 벼는 경북대학교 LMO 격리포장 내에서 재배되었고, 일반 벼는 경상북도 군위군 효령면 화계리에서 재배되었다. 채집방법으로 육안 조사, 포충망 조사, 유아등 트랩, 끈끈이 트랩을 이용하였다. 5년 동안 사천시에서 총 15목 123과 464종 37,941개체가 채집되었고, 2년 동안 군위군에서 총 13목 111과 366종 10,030개체가 채집되었다. 다년간 LMO포장과 일반포장을 비교해본 결과 매년 상위 주요 목은 우점 순서를 제외하면 거의 같은 목이 나타나는 것을 보아 아직까지는 LMO포장과 일반포장의 차이가 뚜렷하게 나타나지 않았다고 사료된다. 그리고 LMO포장과 일반포장 사이의 유사도 지수와 일반포장 사이의 유사도 지수가 차이가 없는 것으로 보아 LMO로 인한 차이가 아닌 환경에 의한 차이로 사료된다.

형질전환작물(Living Genetically Modified Organism; LMO)은 작물을 소비하는 생물의 문제와 더불어 농업생태계에 어떠한 영향을 미치는 지가 주요 관심사로 부각되고 있다. 다년간 형질전환작물이 재배되었던 지역과 비변형 작물 재배지역의 곤충상을 조사하는 것은 각 재배지역의 환경변화를 간접적으로 알 수 있으며, 이를 통해 환경위해성을 평가할 수 있다. 본 연구에서는 2018년 7월부터 같은 해 9월까지 경상남도 사천에 위치한 벼 재배지에서 내충성유전자 변형벼와 일반벼의 발달시기별 곤충상을 조사하고 종다양도 지수, 종균등도 지수, 종풍부도 및 유사도 분석을 포함한 군집분석을 실시하여 유전자변형작물의 환경 및 생태계 위험도 평가를 위한 기초정보를 제공한다.

Bacillus thuringiensis (Bt) produces a variety of insecticidal crystal proteins and widely used as one of the most successful biological control agents. Recently, studies that introduce cry genes into crops to create pest resistance have made much progress, and the total area of land planted with Bt crops has increased substantially. In this study, pest resistance of 8 transgenic Bt rice events with a synthetic cry1Ac gene linked to rice rbcS-tp sequence were assessed under laboratory conditions. Bioassays were performed against Cnaphalocrocis medinalis, which is a significant pest of rice in Asia. C. medinalis larvae were shown to be susceptible to all eight events, even though there were differences between the causes of death. The results differed between developmental stages of the larvae, despite the fact that all 8 events led to high mortalities. These results may be a significant foundation for the evaluation of improved transgenic Bt rice.

Human tissue-type plasminogen activator (t-PA) is responsible for fibrin-specific plasminogen activation and plays a key role in fibrinolysis thereby aiding breakdown of blood clots in the vasculature. In the present study, in order to develop a system for production of recombinant st-PA and t- PAHis6 proteins in transgenic rice seeds, a DNA fragment encoding t-PA gene was selected and cloned to a plant binary vector (pMJ21) harboring a rice GluB1 promoter, an N-terminal signal peptide of the rice glutelin B1 protein and a Pin II terminator. The constructed plasmid was transformed into Agrobacterium tumefaciens LBA4404 (pSB1) to facilitate introduction into rice callus. The insertion of the st-PA and t-PAHis6 genes into the genome of transgenic rice seeds and their transcripts were confirmed using PCR, and Southern blot as well as RT-PCR, respectively. The highest level of recombinant st-PA expression as determined by enzyme-linked immunosorbent assay (ELISA) was found to be 2,916 ng/total soluble protein (mg) in transgenic rice seeds. The amount of recombinant proteins expressed in transgenic plants was estimated to range from 634 ~ 2,916 ng/TSP mg (st-PA) and 925 ~ 2,640 ng/TSP mg(t- PAHis6), respectively. Immuno-blot analysis of transgenic rice seeds revealed single bands of approximately 68-kDa representing recombinant st-PA and t-PAHis6 proteins. These results demonstrate the expression and in vivo activity of recombinant st-PA and t-PAHis6 in transgenic rice seeds. This study is a promising endeavor for production of recombinant pharmaceutical proteins using rice seed system.

농업환경의 급속한 변화에 의한 농업생명공학과 GM작물의증가는 인간의 건강과 자연환경에 대한 깊은 관심을 초래하였고, 이에 대한 환경위험 여부를 확인하기 위해 GM작물의 안전성 평가가 필수 요소가 되었다. 이에 본 연구는 가뭄저항성(Agb0103)벼와 일미벼의 잉어(Carp, Cyprinus carpio)와 미꾸리(Loach, Misgurnus anguillicaudatus)에 대한 급성독성시험을 평가한 결과 48시간과 96시간의 LC50은 1,000 mg/L이상으로 분석되었다. 48시간과 96시간의 무영향농도는 1,000 mg/L이었다. 잉어와 미꾸리의 급성독성 시험기간 중 가뭄저항성(Agb0103)벼와 일미벼 간의 체중, 전장, 수온, DO 및 pH에대한 유의적인 차이는 없었다.

해충저항성 Bt벼와 낙동벼의 미꾸리(Misgurnus anguillicaudatus) 와 잉어(Cyprinus carpio)에 대한 급성독성시험을 실시한 결과 48시간 및 96시간-LC50은 1,000mg/L 이상으로 나타났다. 48시간 및 96시간 무영향농도(NOEC)는 1,000mg/L이었다. 급성독성 시험기간 중 해충저항성 Bt벼와 낙동벼간의 pH, DO, 수온, 체중 및 전장에 대한 유의적인 결과는 나타나지 않았다.

We assessed the environmental risk of herbicide resistant transgenic rice (Protox) on non-target herbivore, grasshoppers (Oxya japonica japonica Thunberg). We conducted life-history experiments of grasshoppers with measuring their body weight, body length, eating amount, and feces amount between non-transgenic rice (nTR; Dongjin rice) and transgenic rice (TR; Protox rice) under laboratory conditions (Temp. 25Ð, R.H. 50-70%, Photoperiod L16:D8) in 2007. The growth of grasshoppers appeared to increase at each measuring date. We also compared the growth rate of grasshoppers between nTR and TR to examine the transgenic impact on the herbivore and we found there was no statistically signifi cant difference between the two plant types (P>0.05). We found that body weight and body length for grasshoppers were highly correlated at each of the two types of plants, nTR (0.962) and TR (0.960). The correlation of eating amount and feces amount of grasshoppers were higher nTR (0.830) than TR (0.782). The energy effi ciency of the grasshopper was not a signifi cant between nTR and TR (P> 0.05). But the molt timing of the grasshoppers for TR difference was faster than for nTR. Conclusively life-history of the grasshoppers but molt timing was not a signifi cant difference between nTR and TR. Therefore, we could conclude there was not any environment risk on herbivore from our result.

To increase thennotolerance of forage crops, transgenic rice plants as a model for transformation of monocots were generated. A cDNA encoding the chloroplast-localized small heat shock protein (small HSP) of rice, Oshsp21, was introduced into rice plants

Antigen production in plant is a safe and effective strategy for vaccine development. In this study, rice transformants were developed for oral vaccine against pigs diarrhea disease. DNA cassette composed with the cholera toxin subunit B (CTB) connected to the 987P-fasG, for stimulating a strong oral immune response, was introduced to rice through Agrobacterium mediated genetic transformation. Copy number analysis by TaqMan real-time PCR for transgenes revealed that transgene of 1 to 8 copies have been introduced into T1 and T2 rice seeds. The expression level of mRNA in the transformants T1 and T2 generations were up to 35 times higher than the reference value in the result of analysis by Quantitative real time-PCR. In addition, the callus cultured from rice transformants was confirmed that the introduced gene has been maintained till 9-month subculture duration. The amount of mRNA expression value was also confirmed in callus, which was maintained above 2.6 times compared with that of the standard control for a long time. These results provide that the introduced antigen for plant-based vaccine against bacterial diarrhea disease can be maintained in the callus as well as in the transgenic plant and suggest that the callus culture of plant transformant will be an effective way to obtain a plant-derived edible vaccine.

A variety of genetically modified (GM) crops have been developed in Korea. In these crops, the resveratrol-enriched transgenic rice plant (Agb0102) has moved ahead to generate the dossier for regulatory review process required for commercialization of GM crop. The resveratrol-enriched transgenic rice plant could be released to farmers for cultivation after national regulators have determined that it is safe for the environment and human health. Here, we developed a PCR-based DNA marker based on flanking sequences of transgene for the discrimination of resveratrol-enriched transgenic rice plant. This DNA markers will be useful for identifying of resveratrol-enriched transgenic rice plant, and can also be used to estimate transgene movement occurred by pollen transfer or seed distribution. Moreover, it is helpful for prompt screening of a homozygote-transgenic progeny in the breeding program.

In this study, we compared disease incidence rate and phyllosphere microbial community between drought resistance transgenic rice (Agb0103) and non-transgenic Ilmi (NGM) during 2011-2014 to examine an environmental risk assessment of drought resistance transgenic rice (Agb0103). As the results, major diseases such as sheath blight, brown spot, leaf blast and false smut were occurred, however, there were no significant disease incidence rate between Agb0103 and NGM. As the results of counting bacterial and fungal viable cell, the colonies were increased or decreased which affected by environmental conditions, however there were no differences between Agb0103 and NGM. Also unweighted pair-group method with arithmetic averaging (UPGMA) analysis based on polymerase chain reaction with denaturing gel electrophoresis (PCR-DGGE) revealed that DGGE band pattern of bacterial and fungal communities were clustered by each month and there were no differences between Agb0103 and NGM. Furthermore, isolated casual agents causing sheath blight and brown spot were collected from Agb0103 and NGM, and they revealed that each of pathogens were no differences in morphology and pathogenicity. Therefore, our results suggested that Agb0103 showed no differences in disease incidence rate, characteristic of pathogens and phyllosphere community with NGM. In this way, it can be assumed that transgenic rice Agb0103 could not influence phyllosphere microorganism community and environmental conditions.

The selectable marker-free rice plants containing mcry1Ac insecticidal gene isolated from Bacillus thuringiensis (Bt) were generated using a non-selection approach by Agrobacterium tumefaciens-mediated transformation. The nutritional composition of two lines of transgenic rice plants (RTB5 and RTB11) was compared with that of its non-transgenic counterpart. The results showed that, except for small differences in dietary fiber and some minerals, there was no significant difference between transgenic rice and conventional counterpart variety with respect to their nutrient composition. Most of measured levels of nutrients were within the range of values reported for other commercial cultivars, showing substantial equivalency. Therefore, the insertion of transgenes did not affect the nutritional composition of transgenic RTB5 and RTB11 rice grains.

A variety of genetically modified (GM) crops have been developed in Korea. In these crops, the resveratrol-enriched transgenic rice plant has moved ahead to generate the dossier for regulatory review process required for commercialization of GM crop. The resveratrol-enriched transgenic rice plant could be released to farmers for cultivation after national regulators have determined that it is safe for the environment and human health. Here we developed a PCR-based DNA marker based on flanking sequences of transgene for the discrimination of zygosity in resveratrol-enriched transgenic rice plant. This DNA marker will be useful for identifying of resveratrol-enriched transgenic rice plant, and can also be use to estimate transgene movement occurred by pollen transfer or seed distribution.

The β-carotene biofortified transgenic rice was developed by transforming rice cv. Nakdongbyeo with phytoene synthase (Psy) and carotene desaturase (Crt I) genes isolated from Capsicum and Pantoea. The aim of this study was to perform molecular characterization of rice transformants of T5-T7 generation harboring Psy and Ctr I genes driven by endosperm specific globulin promoter for biosafety evaluation of β-carotene biofortified transgenic rice. The structure and sequence of T-DNA in the transformation vector and the insertion sites, flanking sequences and generational stability of inserted T-DNA in transgenic rice lines were analyzed. The transformation vector consisted of right border, MAR gene, carotenogenic genes unit, herbicide resistance selectable marker unit, MAR gene and left border in sequential order. T-DNA was introduced at the position of 30,363,938-30,363,973 bp of chromosome No. 2 by adaptor-ligation PCR. Stable integration of T-DNA and stable expression of bar gene was confirmed in T5 to T7 generations. It was also confirmed that the backbone DNA of transformation vector containing antibacterial gene was not present in the genome of β-carotene biofortified transgenic rice. HPLC analysis confirmed that carotenoids were consistently detected through T5-T7 generations.