무궁화 품종 '난파'의 효율적인 재분화 체계 확립

손지희,선현진,강홍규,서석철,이효연,Son, Ji-Hi,Sun, Hyeon-Jin,Kang, Hong-Gyu,Suh, Seok-Chul,Lee, Hyo-Yeon 한국식물생명공학회 2019 식물생명공학회지 Vol.46 No.4

Hibiscus syriacus L., the national flower of Korea, is a commonly grown ornamental species found in parks, gardens and along roadsides in Korea. This study sought to establish a plant regeneration system of H. syriacus 'Nanpa' using mature leaves as an explant. Sterilized mature leaf explants were cultured on modified MS medium with combinations of NAA and BAP at various concentrations for 6 weeks. Among the various media evaluated, modified MS media containing 0.46 mg/L NAA and 0.5 mg/L BAP (CI-1) or 0.92 mg/L NAA and 1 mg/L BAP (CI-4) were the most effective for callus formation. Whitish-yellow calluses were observed on CI-1 medium, while green calluses formed on CI-4 medium. The whitish-yellow callus derived from CI-1 medium showed a higher frequency of shoot induction than the green callus derived from CI-4 on modified MS medium containing 0.5 mg/L BAP. Among the various media evaluated in this study, modified MS medium containing 0.46 mg/L NAA and 0.5 mg/L BAP was the most optimal for shoot-forming callus production. Our findings show that mature leaves of H. syriacus 'Nanpa' can be used for in vitro plant regeneration, and the regeneration system described here may be a powerful tool for molecular breeding of H. syriacus 'Nanpa' in the future.

제초제저항성 GM 잔디에서 발현된 PAT 단백질의 알레르겐 유발 가능성 및 독성 평가

정혜린,선현진,강지남,강홍규,이효연,Jeong, Hye-Rin,Sun, Hyeon-Jin,Kang, Ji-Nam,Kang, Hong-Gyu,Lee, Hyo-Yeon 한국식물생명공학회 2020 JOURNAL OF PLANT BIOTECHNOLOGY Vol.47 No.4

본 연구는 제초제저항성 GM 들잔디에서 발현된 PAT 단백질의 잠재적인 알레르기성 및 독성을 평가하기 위해 수행되었다. PAT 단백질의 in silico 분석에서 PAT 단백질은 알려진 알레르겐 또는 독성 단백질과 유사성을 보이지 않았다. PAT 단백질은 80개의 아미노산으로 이루어진 절편에 걸쳐 기지의 알레르겐과 35% 미만의 아미노산 서열 상동성을 보였고, 기지의 알레르겐과 연속한 8개의 아미노산에서 일치하는 서열도 확인되지 않았다. 또한 제초제저항성 GM 들잔디에서 발현된 PAT 단백질은 펩신 존재 하의 인공위액에서 매우 빠르게 분해되었고, GM 들잔디에서 발현된 PAT 단백질은 번역 후 수식(glycosylation)도 일어나지 않았음이 확인되었다. 경구투여 독성 시험에서는 체중 1 kg 당 4,000 mg 을 투여한 실험구에서도 PAT 단백질 투여 후 마우스에서 사망이나 독성 영향은 관찰되지 않았다. 이들 결과는 제초제저항성 GM 들잔디에서 발현된 PAT 단백질이 잠재적 알레르겐이나 독소로 작용할 가능성이 없음을 시사한다. This study aimed to evaluate the potential allergenicity and oral toxicity of the phosphinothricin acetyltransferase (PAT) protein expressed in Zoysia japonica, a herbicide-tolerant genetically modified (GM) zoysiagrass. In silico analysis of PAT showed no similarities with any known allergenic or toxic proteins, with <35% amino acid sequence homology with known allergens across a length of 80 amino acids and no continuous eight amino acid identity with known allergens. The PAT protein expressed in Z. japonica degraded very rapidly in the simulated gastric fluid in the presence of pepsin, and, no glycosylation of PAT was observed. The oral toxicity test revealed no mortality or toxic effect in mice following PAT administration at 4,000 mg/kg body weight. Our findings indicate that the PAT protein expressed in Zoysia japonica does not exhibit allergenic or toxic properties.

들잔디로부터 β-1,3-glucanase 유전자의 클로닝 및 특성분석

강소미,강홍규,선현진,양대화,권용익,고석민,이효연,Kang, So-Mi,Kang, Hong-Gyu,Sun, Hyeon-Jin,Yang, Dae-Hwa,Kwon, Yong-Ik,Ko, Suk-Min,Lee, Hyo-Yeon 한국식물생명공학회 2016 식물생명공학회지 Vol.43 No.4

한국형 잔디에서는 다른 병에 비해 진전 속도가 빠르고 주로 뿌리에서부터 발병하여 잔디를 고사시키고 발병 후 구제하기 매우 어려운 라이족토니아잎마름병(라지패취)이 큰 문제로 대두되고 있다. 라이족토니아잎마름병(라지패취)은 Rhizoctonia solani AG2-2 (IV)병원균에 의해 발생하는데, 이 병원균에 강한 내병성 들잔디를 개발하기 위해 식물방어반응에 중요한 역할을 하는 것으로 알려진 PR-Protein 중 하나인 ${\beta}-1,3-glucanase$를 들잔디로부터 클로닝 하였다. ${\beta}-1,3-glucanase$는 바이러스나 균의 감염으로 인해 식물조직이 과민반응을 일으킬 때 세포내에서 생성되고 세포 외로 분비되어 세포 사이 공간에서 주로 병원균 저항성기능을 하는 것으로 알려져 있다. ${\beta}-1,3-glucanase$ 단자엽식물 중 내병성에 대한 연구가 되어있는 옥수수, 밀, 보리, 벼의 염기서열에서 공통으로 보존되어 있는 부분을 이용해 degenerate PCR을 수행하고 얻어낸 sequence를 통해 Full-length의 cDNA를 클로닝 하였다. E.coli overexpression을 수행하여 목표 단백질을 대량 정제하여 in vitro 활성 측정 및 항균테스트를 진행하였다. 또한, ZjGlu1 유전자의 기능을 해석하기 위해 각각의 유전자를 도입한 식물형질전환용 벡터를 제작하여 잔디 형질전환체 제작을 하였다. ZjGlu1 단백질을 이용하여 9개의 균주에 대해 항균활성 테스트를 진행 한 결과 R. cerealis, F. culmorum, R.solani AG-1 (1B), T. atroviride 에서 항균활성을 보였으며, 형질전환체를 이용해 18s 유전자의 발현량을 상대로 한 각 유전자의 기관별 발현량은 크게 차이없이 모든기관에 발현되는 것을 확인할 수 있었다. Rhizoctonia leaf blight (large patch) has become a serious problem in Korean lawn grass, which is extremely hard to treat and develops mostly from the roots of lawn grass to wither it away. Rhizoctonia leaf blight (large patch) is caused by Rhizoctonia solani AG2-2 (IV). To develop zoysia japonica with strong disease tolerance against this pathogenic bacterium, ${\beta}-1,3-glucanase$ was cloned from zoysia japonica, which is one of the PR-Proteins known to play a critical role in plant defense reaction. ${\beta}-1,3-glucanase$ is known to be generated within the cells when plant tissues have a hypersensitive reaction due to virus or bacterium infection and secreted outside the cells to play mainly the function of resistance against pathogenic bacteria in the space between the cells. This study utilized the commonly preserved part in the sequence of corn, wheat, barley, and rice which had been researched for their disease tolerance among the ${\beta}-1,3-glucanase$ monocotyledonous plants. Based on the part, degenerate PCR was performed to find out the sequence and full-length cDNA was cloned. E.coli over-expression was conducted in this study to mass purify target protein and implement in vitro activation measurement and antibacterial test. In addition, to interpret the functions of ZjGlu1 gene, each gene-incorporating plant transformation vectors were produced to make lawn grass transformant. Based on ZjGlu1 protein, antibacterial activity test was conducted on 9 strains. As a result, R. cerealis, F. culmorum, R.solani AG-1 (1B), and T. atroviride were found to have antibacterial activity. The gene-specific expression amount in each organ showed no huge difference in the organs based upon the transformant and against 18s gene expression amount.

다양한 스트레스에 유도되는 들잔디 ZjWRKY3, ZjWRKY7의 분석

김우남,송인자,강홍규,선현진,양대화,이용억,권용익,이효연,Kim, Woo-Nam,Song, In-Ja,Kang, Hong-Gyu,Sun, Hyeon-Jin,Yang, Dae-Hwa,Lee, Yong-Eok,Kwon, Yong-Ik,Lee, Hyo-Yeon 한국식물생명공학회 2017 식물생명공학회지 Vol.44 No.3

식용작물, 사료, 잔디를 포함하는 모든 작물은 건조, 염, 저온, 고온 등의 여러 가지 환경스트레스의 영향을 빈번히 받기 때문에 작물의 생산성이 떨어지게 된다. 식물은 환경스트레스 상황에서 스스로 벗어날 수 없다. 따라서 식물은 환경 스트레스를 극복하는 방향으로 진화하였다. ARF, ABI3, NAC, HSF, WRKY 같은 환경 스트레스에 반응하는 유전자들이 식물에서 보고되었다. 이 유전자들은 환경스트레스에 반응하는 전사인자로, 식물의 스트레스반응 경로에 연관되어 있다. OsWRKY76의 경우에는 저온 및 병원균에 대한 내성을 증가시켰고, AtWRKY28 의 경우 여러 가지 환경스트레스에 관련이 있는 것으로 보고되었다. 들잔디는 정원이나 골프코스에서 가장 흔하게 사용되는 잔디이다. 하지만 들잔디에서는 아직 WRKY 유전자가 알려지지 않았다. 본 연구에서는 들잔디로부터 1개의 WRKY domain을 포함하는 ZjWRKY3, ZjWRKY7 를 분리하였다. ZjWRKY3과 ZjWRKY7은 저온, 건조, 염 스트레스에 발현이 증가하였다. 들잔디의 갈색퍼짐병을 일으키는 R. solani의 감염이 ZjWRKY3과 ZjWRKY7의 발현을 증가시켰다. 또한 ZjWRKY3, ZjWRKY7이 Zjchi 유전자 promoter의 W-box에 결합하여 전사를 조절한다는 사실을 확인 하였다. 따라서 ZjWRKY3, ZjWRKY7 유전자는 전사인자로서 환경스트레스 및 병원균 관련 하위 유전자들을 조절할 것으로 예상된다. Many crops including cereals, tuber crops, feeds, and turf grasses are often damaged by various environmental stresses such as drought, salt, cold, and high temperature, causing the reduction of their productivity. Plants are sessile and cannot escape from environmental stresses. Thus, plants evolve in the direction of overcoming the environmental stresses. Some plant genes such as ARF, ABI3, NAC, HSF, and WRKY are known to respond to environmental stresses as they transcriptionally regulate the stress response pathways. For example, the OsWRKY76 gene contributes to the enhanced resistance to low temperatures and pathogenic infections. The AtWRKY28 also plays a role in environmental stresses. Zoysiagrass (Zoysia japonica Steud.) is popularly grown for gardens and golf courses. However, the function of the WRKY gene, another environmental stress-related gene, is not known in zoysiagrass. In this study, the ZjWRKY3 and ZjWRKY7 genes with one shared WRKY domain have been isolated in zoysiagrass. The expression of these genes increased in response to low temperature, drought, and salt stresses. Furthermore, the infection of the brown patch-causing Rhozoctonia solani induced the expression of ZjWRKY3 and ZjWRKY7. The corresponding proteins bind to the W-box of the Zjchi promoter, possibly regulating their transcriptions. The researchers suggest that the ZjWRKY3 and ZjWRKY7 genes transcriptionally regulate abiotic and biotic stress related downstream genes.

저온에서 녹기연장의 한국잔디 ‘한라그린 7’ 품종 개발

양대화(Dae-Hwa Yang),선현진(Hyeon-Jin Sun),정옥철(Ok-Cheol Jeong),강홍규(Hong-Gyu Kang),송인자(In-Ja Song),진일두(Il-Doo Jin),강시용(Si-Yong Kang),이효연(Hyo-Yeon Lee) 한국육종학회 2021 한국육종학회지 Vol.53 No.4

The ‘Halla Green 7’ cultivar is a hybrid of Zoysia matrella and Zoysia japonica. The inflorescences of Z. matrella lines werecollected from Jeju Island near the border between Z. matrella and Z. japonica wild plants, respectively. After their harvest, the seeds weresown and various interspecific lines were obtained. In 2021, a line with greening period extending phenotype during the low temperaturesof winter was registered under the cultivar name of ‘Halla Green 7’ (Registration No. 245). The main morphological characteristics of the‘Halla Green 7’ variety are as follows: genetically short length of plant (12.2±0.58 cm), very wide angle of leaf (68.6°), short length ofleaf blade (8.38±0.37 cm), medium-leaf (3.29±0.09 mm), and very low leaf sheath (2.18±0.19 cm). The short length of leaf sheath makesit easy to manage the turf grass with low height. A very wide leaf angle has a high photosynthetic efficiency. The ‘Halla Green 7’ cultivaris morphologically classified as Z. matrella × Z. japonica hybrid line by medium-width of leaf blade and the presence of trichomes on theleaf surface. The colors of the leaf blade, leaf sheath, and stolon are green, light green, and light purple, respectively. During the low temperatureperiod of winter, chlorophyll content of the leaves of ‘Halla Green 7’ shows a 10.1-fold from that of the control Z. japonia ‘Yaji’ and about4.2-fold from that of the control Z. matrella Zm-6. This greening period extending phenotype is expected to contribute to the industrializationof zoysiagrass (Registration No. 245).

첩포시험을 이용한 유전자 변형 들잔디의 접촉피부염 인체위해성 검정

이재천 ( Jae Chun Lee ),선현진 ( Hyeon Jin Sun ),이효연 ( Hyo Yeon Lee ) 대한천식알레르기학회 2015 Allergy Asthma & Respiratory Disease Vol.3 No.2

Purpose: Genetic modification technique is widely used in plants for improving crop yields and qualities, and reducing pesticide use. Zoysia japonica is one of the widely planted grasses for lawning. Pollenless herbicide-tolerant genetically modified (GM) grasses (JG21-MS1 and JG21-MS2) were developed under surveillance for possible biohazard. Grasses may cause allergic contact dermatitis with direct contact on human skin. Patch testing with allergens was adopted to compare the incidences of allergic contact dermati￢tis to GM grass leaves with those of wild-type grass. Methods: Patch testing with controls and leaves of wild-type and GM grasses was performed in individuals with informed consent. Results: Ninety-seven individuals (mean age, 32±8 years; 48% males) were enrolled. For nickel sulfate as a positive control, 38 sub￢jects (39.2%) were positive. For GM grasses, 10 (10.3%) showed positive patch test results and 11 (11.3%), for wild-type grass, the proportions of which were similar among the tested grasses. Conclusion: JG21-MS1 and JG21-MS2 are not more biohazardous than wild-type grass in the risk of allergic contact dermatitis.(Allergy Asthma Respir Dis 2015;3:134-138)

왜성 들잔디 신품종 ‘한라그린2’의 개발

송인자(In-Ja Song),선현진(Hyeon-Jin Sun),정옥철(Ok-Cheol Jeong),양대화(Dae-Hwa Yang),진일두(Il-Doo Jin),강홍규(Hong-Gyu Kang),고석민(Suk-Min Ko),권용익(Yong-Ik Kwon),배태웅(Tae-Woong Bae),송필순(Pill-Soon Song),이효연(Hyo-Yeon Lee) 한국육종학회 2017 한국육종학회지 Vol.49 No.1

This study was carried out to develop new zoysiagrass (Zoysia japonica Steud.) cultivar ‘Halla Green 2’ (Grant number: No. 118). To develop a zoysiagrass cultivar with dwarfism by using the mutation breeding method, the wild type control Gosan plants were irradiated using a 30 Gy gamma ray source in 2010. Dwarf mutants were selected from the mutated grasses in successive generations. Dwarf mutant lines were identified and a new zoysiagrass variety Halla Green 2 was developed. The plant height of Halla Green 2 was 3.4 and 1.8 times lower than that of Gosan and Zenith, respectively. This cultivar has dwarf characteristics such as shorter sheath, shorter leaf blade, shorter flag leaf, and shorter third internode of stolon compared to those of Gosan and Zenith. Additionally, the sheaths and leaf blades color of Gosan, Zenith and Halla Green 2 were all light green, whereas their stolons were purple, yellow-green and yellow green, respectively. Trichomes(hairs) were visible on both adaxial and abaxial surfaces of the Gosan leaves, whereas only on the adaxial side of the Zenith and Halla Green 2 leaves. The Halla Green 2 grass showed distinguishable morphological traits compared to those of wild type Gosan and Zenith.

FT-IR스펙트럼 데이터의 다변량통계분석 기반 들잔디와 갯잔디의 대사체 수준 신속 식별 체계

양대화,안명숙,정옥철,송인자,고석민,전예인,강홍규,선현진,권용익,김석원,이효연,Yang, Dae-Hwa,Ahn, Myung Suk,Jeong, Ok-Cheol,Song, In-Ja,Ko, Suk-Min,Jeon, Ye-In,Kang, Hong-Gyu,Sun, Hyeon-Jin,Kwon, Yong-Ik,Kim, Suk Weon,Lee, Hyo-Yeon 한국식물생명공학회 2016 식물생명공학회지 Vol.43 No.2

본 연구에서는 FT-IR 스펙트럼 분석을 통해 한국에서 자생하는 Zoysia 속인 들잔디(Zoysia japonica)와 갯잔디(Zoysia sinica)의 전세포추출 시료로부터 대사체 수준에서 신속한 식별체계를 확립하고자 하였다. 이를 위해 기준라인으로 분자마커를 이용해 동정이 완료된 들잔디와 갯잔디 시료를 FT-IR 분석에 사용하였으며, 제주도와 전라도에서 수집된 미동정 잔디들을 기준라인과 비교분석하기 위해 FT-IR 분석에 사용하였다. 기준라인 들잔디와 갯잔디 시료로부터 확보된 FT-IR 스펙트럼 데이터의 PCA (principal component analysis)와 PLS-DA (partial least square discriminant analysis) 분석 결과 각 기준라인은 들잔디 및 갯잔디 종에 따라 뚜렷하게 식별되었다. 들잔디와 갯잔디 시료 사이에서 가장 큰 PC loading value값을 보인 부위는 $1,100{\sim}950cm^{-1}$였다. 이 부위는 carbohydrates 계열의 화합물들의 질적, 양적 정보를 반영하는 부위로 이 계열의 화합물의 양적, 질적 차이가 들잔디, 갯잔디의 대사체 수준 구분에 중요한 역할을 하고 있음을 알 수 있었다. 기준라인 들잔디와 갯잔디 시료집단에 미동정된 잔디 시료 집단을 추가하여 PCA와 PLS-DA 분석한 결과, 일차적으로 들잔디와 갯잔디로 구분이 이루어졌으며 미동정 집단은 모두 들잔디 그룹내에 분포하였다. 특히, HCA (hierarchical clustering analysis) dendrogram 분석 결과에서 동정 및 미동정 들잔디 시료들은 모두 수집지 특성에 따라 국내 국립공원의 고산지대와 국내 섬지역 해안가의 저지대로 별도의 소그룹을 형성하였다. 따라서, 본 연구 결과에서 확립된 FT-IR 스펙트럼 분석법은 한국 전역에 자생하는 들잔디와 갯잔디의 신속한 종 식별뿐만 아니라 수집 지역의 특성에 따라 대사체 수준에서의 유연관계를 규명하는데 활용 가능할 것으로 기대된다. This study aims to establish a system for the rapid discrimination of Zoysia species using metabolite fingerprinting of FT-IR spectroscopy combined with multivariate analysis. Whole cell extracts from leaves of 19 identified Zoysia japonica, 6 identified Zoysia sinica, and 38 different unidentified Zoysia species were subjected to Fourier transform infrared spectroscopy (FT-IR). PCA (principle component analysis) and PLS-DA (partial least square discriminant analysis) from FT-IR spectral data successfully divided the 25 identified turf grasses into two groups, representing good agreement with species identification using molecular markers. PC (principal component) loading values show that the $1,100{\sim}950cm^{-1}$ region of the FT-IR spectra are important for the discrimination of Zoysia species. A dendrogram based on hierarchical clustering analysis (HCA) from the PCA and PLS-DA data of turf grasses showed that turf grass samples were divided into Zoysia japonica and Zoysia sinica in a species-dependent manner. PCA and PLS-DA from FT-IR spectral data of Zoysia species identified and unidentified by molecular markers successfully divided the 49 turf grasses into Z. japonica and Z. sinica. In particular, PLS-DA and the HCA dendrogram could mostly discriminate the 47 Z. japonica grasses into two groups depending on their origins (mountainous areas and island area). Considering these results, we suggest that FT-IR fingerprinting combined with multivariate analysis could be applied to discriminate between Zoysia species as well as their geographical origins of various Zoysia species.

한국잔디 ‘한라그린 10’ 품종의 개발

양대화(Dae-Hwa Yang),정옥철(Ok-Cheol Jeong),선현진(Hyeon-Jin Sun),강홍규(Hong-Gyu Kang),진일두(Il-Doo Jin),이효연(Hyo-Yeon Lee) 한국육종학회 2021 한국육종학회지 Vol.53 No.4

This superior zoysiagrass line was selected from the zoysiagrass genetic resources collected in the Republic of Korea. Inflorescencesof Zoysia matrella lines were collected near the border between wild Zoysia japonica and wild Z. matrella on Jeju Island. Various Z. matrella× Z. japonica lines were obtained using the seeds from the collected inflorescences. In 2021, one of the lines that extended the greeningperiod during the low temperatures of winter was registered under the name of ‘Halla Green 10’. The main morphological characteristicsof this cultivar, which were analyzed using the test guidelines of the Korea Forest Service, are as follows: short length of plant (12.01±0.57cm), short length of leaf blade (8.83±0.68 cm), and short length of leaf sheath (1.71±0.20 cm). The short length of leaf sheath makes iteasy to manage the turf grass with low height. Due to the presence of trichomes on the leaf surface and medium-leaf type (leaf blade width:3.0±0.16 mm), ‘Halla Green 10’ is morphologically classified as Z. matrella × Z. japonica. As the leaf angle is very wide at 77.7°, it hasa high photosynthetic efficiency. The colors of the leaf sheath, leaf blade, and stolon are light green, green, and light purple, respectively. In particular, during the low temperature period of winter, the chlorophyll content of the leaves of ‘Halla Green 10’ shows about 7.5-foldfrom that of the control Z. japonia ‘Yaji’ and about 3.2-fold increase from that of the control Zm-6. Extending the greening period phenotypeis expected to be commercially useful (Registration No. 246).

웅성불임 및 왜성형질의 제초제저항성 들잔디(zoysia japonica Steud.)의 판별기술 개발

이범규(Bum kyu Lee),강홍규(Hong-Gyu Kang),라누리(Nu ri Ra),선현진(Hyeon-Jin Sun),권용익(Yong-Ik Kwon),송인자(In-Ja Song),김창기(Chang-Gi Kim),류태훈(Tae-Hun Ryu),박기웅(Kee Woong Park),이효연(Hyo-Yeon Lee) 충남대학교 농업과학연구소 2014 농업과학연구 Vol.41 No.3

The cultivation area and use of genetically modified (GM) crops have been increased continuously over the world. Concerns about the potential risks of GM crops are also increasing. Safe management for the development and production of GM crops is required according to Living Modified Organism Act in Korea. Planning about the methods, duration, and frequency of environmental monitoring is also required for commercial use of GM crops. GM Zoysia japonica Steud. (event name: JG21) expressing resistance to glufosinate-ammonium has been generated previously. By using gamma ray treatment to JG21 we also developed male sterility and dwarf Z. japonica (event name: JG21-MS). The objective of this study was to establish the monitoring system for environment release of JG21-MS. In this study we extracted RNA from JG21 and JG21-MS and conducted RAPD (random amplified polymorphic DNA) method to distinguish JG21 and JG21-MS.