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Glyphosate에 대한 옥수수 반응의 개선된 검정방법
김진석,이병회,김소희,민석기,최정섭,Kim, Jin-Seog,Lee, Byung-Hoi,Kim, So-Hee,Min, Suk-Ki,Choi, Jung-Sup 한국식물생명공학회 2006 식물생명공학회지 Vol.33 No.1
본 연구에서는 옥수수를 대상으로 glyphosate에 대한 여러 가지 생리적 반응을 검토한 후, glyphosate 저항성 평가에 활용될 수 있는 보다 개선된 방법 두 가지를 확립하였다. 한 가지 방법은 옥수수 제3엽 상단에 약제를 국소 처리한 다음, 처리 후 3일째에 약제처리 되지 않았던 제4엽의 신장 정도를 조사하는 것이다 (전식물체-엽생장 검정). 이 경우 glyphosate $50-1,600{\mu}g/mL$ 범위에서 농도가 증가됨에 따라 엽 생장이 억제되었으며, $1,600{\mu}g/mL$ 농도에서의 생장 억제율은 무처리 대비 55.5%였다. 다른 한 가지 방법은 옥수수 제3본엽의 엽절편 ($4{\times}4mm$) 4개씩을 $200{\mu}L$의 시험용액이 담긴 48 well plate에 치상한 후 $25^{\circ}C$ 연속 명조건에 24시간동안 배양하여 shikimate 축적량을 조사하는 것이다 (엽절편-shikimate 축적 검정). 이 경우 시험용액에 0.33% sucrose를 가하면 무첨가에 비해 약3-4배 정도의 shikimate 축적 증가가 관찰되었고 glyphosate $2-8{\mu}g/mL$ 농도범위에서 직선적 증가반응을 나타내어 기존방법 (Shaner et al. 2005)보다 개선된 특징을 보였다. 본 방법들은 glyphosate 저항성 옥수수를 창출할 때 또는 저항성 유전자의 타 식물로의 이동여부와 잡초화된 저항성 옥수수 존재여부를 감별하는데 활용될 수 있을 것이다. 이 때 glyphosate에 대한 저항성 원인이 작용점 EPSPS와 관련이 있는 경우에는 "엽절편-shikimate 축적 검정"이 가장 바람직하고, 저항성 원인이 체내이행 감소 때문일 경우에는 "전식물체-엽생장 검정" 수행이 필요하다. Several methods for determining the response of corn to glyphosate were investigated to provide a fast and reliable method for identifying glyphosate-resistant corn in vivo. Two bioassays were developed. One assay is named 'whole plant / leaf growth assay', in which the herbicide glyphosate is applied on the upper part of 3rd leaf and the growth of herbicide-untreated 4th leaf is measured at 3 day after treatment. in this assay, the leaf growth of conventional corn was inhibited in a dose dependent from 50 to $1600{\mu}g/mL$ of glyphosate and growth inhibition at $1600{\mu}g/mL$ was 55% of untreated control. The assay has the potential to be used especially in the case that the primary cause of glyphosate resistance is related with a reduction of the herbicide translocation. Another assay is named 'leaf segment / shikimate accumulation assay', in which the four excised leaf segments ($4{\times}4mm$) are placed in each well of a 48-well microtiter plate containing $200{\mu}L$ test solution and the amount of shikimate is determined after incubation for 24 h in continuous light at $25^{\circ}C$. In this assay, 0.33% sucrose added to basic test solution enhanced a shikimate accumulation by 3 to 4 times and the shikimate accumulation was linearly occurred from 2 to $8{\mu}g/mL$ of glyphosate, showing an improved response to the method described by Shaner et al. (2005). The leaf segment / shikimate accumulation assay is simple and robust and has the potential to be used as a high throughput assay in the case that the primary cause of glyphosate resistance is related with EPSPS, target site of the herbicide. Taken together, these two assays would be highly useful to initially select the lines obtained after transformation, to investigate the migration of glyphosate-resistant gene into other weeds and to detect a weedy glyphosate-resistant corn in field.
백화옥수수 잎절편을 이용한 안토시아닌 생합성 조절제 탐색용 신규 검정법 확립
김진석,이병회,이정애,오광훈,조광연,Kim, Jin-Seog,Lee, Byung-Hoi,Lee, Jung-Ae,Oh, Kwang-Hoon,Cho, Kwang-Yun 한국식물생명공학회 2003 식물생명공학회지 Vol.30 No.2
This study was carried out to establish a novel bioassay system for screening of compounds affecting aromatic amino acid or anthocyanin biosynthesis through investigating a degree of sucrose-induced anthocyanin formation such as size of plant material, buffer conditions, light intensity and irradiated duration, incubation temp., etc were determined and standard procedure (suitable experimental condition) was set up as follows. The second leaf blade of white corn seeding induced by fluridone treatment were segmented into a size of 5${\times}$5 min. The segments were floated on the solution of 1% sucrose in 1.0mM MES buffer (pH6.0∼6.5) and incubated at 26$^{\circ}C$ for 2days under the continuous light condition(70∼100$\mu$mol m$^{-2}$ s$^{-1}$ ). Anthocyanin in the purpled tissues was extracted with methanol containing 1% HCl and the optical density of the clear supematants was determined at 528mm. Influences of some chemicals were tested using this system. Glyphosate, 5-enolpyruvylshikimate 3-phosphate synthase inhibitor, showed most sensitive response with I$_{50}$ value at 3.3$\mu$M. Dicyclohexylcarbodiimide(DCCD) and parachloromercuribenzenesulfonic acid(PCMBS) had a relatively strong ingibition with I50 value at 7.1$\mu$M and 10.2$\mu$M, respectively. These results show that sucrose-induced anthocyanin formation in white com leaf segment provide a very simple and rapid system for searching new compounds affecting aromatic amino acid or anteocyanin biosynthesis by screening at less than 10$\mu$M.
Superoxide Dismutase와 Ascorbate Peroxidase가 엽록체내로 동시에 과대발현된 형질전환 담배의 제초제들에 대한 항산화 반응
김진석,이병회,권석윤,김윤희,김소희,조광연,Kim Jin-Seog,Lee Byung-Hoi,Kwon Suk-Yoon,Kim Yun-Hee,Kim So-Hee,Cho Kwang-Yun 한국식물생명공학회 2005 식물생명공학회지 Vol.32 No.2
Superoxide dismutase와 ascorbate peroxidase가 동시에 과대발현된 담배(CA)를 가지고 여러 제초제들에 대한 항산화 반응을 조사하였다. 온실조건 실험에서, CA 담배는 PSI 저해제로 알려진 paraquat처리에 대해서 내성이 인정되었고 그 정도는 40% 내외였다. PS II 저해제 (bromoxynil, diuron, bromacil), 엽록소 생합성 저해제 (oxyfluorfen), 카로티노이드 생합성 저해제 (fluridone)와 EPSP synthase 저해제 (glyphosate) 처리에서는 CA와 wild type간의 반응차이가 관찰되지 않았다. Paraquat와 diuron을 이용한 약광 조건의 실험에서도 온실조건의 실험결과와 유사한 정도로 paraquat처리에 대해서만 내성을 나타내었다. 온실조건에서의 엽위별 반응의 경우, 6 - 9 엽기 식물체에 paraquat를 처리하였을 때, 약제처리 당시 위로부터 3 - 4번째 전개되고 있었던 잎이 상대적으로 내성 정도가 높게 나타났다. 한편 paraquat 처리 시에 여러 농도의 ascorbic acid를 혼합할 경우, CA와 wild type 모두에서 비슷한 정도로 paraquat 활성을 경감시켰다. 결론적으로, CuZnSOD/APX의 과대발현은 photosystem I 에서 발생되는 산화스트레스에 대해서만 주로 작용하며, 다른 제초제들에 의해 발생되는 산화적 스트레스에 대해서는 소거 능력이 부족한 것으로 판단되었다. Antioxidative responses of transgenic tobacco plants expressing both superoxide dismutase (SOD) and ascorbate peroxidase (APX) in chloroplasts was investigated with several herbicides. In greenhouse test, tolerance of SOD/APX-overexpressed tobacco (CA) to photosystem (PS) I inhibitor paraquat was increased by about 40%. However, any response differences between CA and wild type (WT) tobacco was not observed in a treatment with PS II inhibitors (bromoxynil, diuron and bromacil), chlorophyll biosynthesis inhibitor(oxyfluorfen), carotenoid biosynthesis inhibitor (fluridone) and 5-enolpyruvylshikimate-3-phosphate (EPSP) synthase inhibitor (glyphosate). This tendency was also similar in the growth chamber test of low light intensity, using paraquat and diuron. That is, increased antioxidant activity of CA was shown only in paraquat treatment. When paraquat was foliar-treated to 6 to 9-leaf stage plant, the third to fourth placed leaf from shoot tip showed relatively higher antioxidant activity. Ascorbate supplemented to paraquat solution alleviated the phytotoxicity with a similar range in both CA and WT. In conclusion, CA specifically responded to oxidative stress induced by paraquat among tested herbicides in a whole plant assay.
갈근에서 분리한 화합물의 $\alpha$-glucosidase와 $\alpha$-amylase 활성 저해 효과
박지희,백목련,이병회,연규환,유시용,김영섭,박상언,홍경식,Park, Jee-Hee,Baek, Mok-Ryeon,Lee, Byung-Hoi,Yon, Gyu-Hwan,Ryu, Shi-Yong,Kim, Young-Sup,Park, Sang-Un,Hong, Kyung-Sik 한국약용작물학회 2009 한국약용작물학회지 Vol.17 No.5
Compounds of isolated from roots extract of Pueraria thunbergiana were tested their inhibitory effects on $\alpha$-glucosidase and $\alpha$-amylase. Inhibitory activity of methylene chloride (MC) fraction and ethyl acetate (EA) fraction against $\alpha$-glucosidase showed more than 60% at a concentration of $500{\mu}g/m{\ell}$. Among the nine compounds tested on $\alpha$-glucosidase, biochanin A, (-)-tuberosin and calycosin from MC fraction and daidzein from EA fraction were stronger inhibitors than acarbose ($IC_{50}=530{\mu}g/m{\ell}$), and their $IC_{50}$ were 9, 144, 328 and $20{\mu}g/m{\ell}$, respectively. Biochanin A and (-)-tuberosin also inhibited $\alpha$-amylase activity as like as acarbose $IC_{50}=20.5{\mu}g/m{\ell}$), and their $IC_{50}$ were 22 and $348{\mu}g/m{\ell}$, respectively. Although daidzein was already known $\alpha$-glucosidase inhibitory effects, it was newly evaluated that biochanin A and (-)-tuberosin inhibited $\alpha$-glucosidase as well as $\alpha$-amylase, and that calycosin did $\alpha$-glucosidase.