들잔디 5-Enolpyruvyl Shikimate 3-Phosphate Synthase(EPSPS) 유전자 클로닝 및 특성 Cloning and Characterization of a 5-Enolpyruvyl Shikimate 3-Phosphate Synthase (EPSPS) Gene from Korean Lawn Grass (Zoysia japonica)원문보기
본 연구에서는 들잔디와 돌연변이체에서 Glyphosate 내성 관련 유전자인 EPSPS를 코딩하는 cDNA를 분리하여, 시퀸스 비교분석과 발현 양상 차이 등에 관하여 조사하였다. 5'/3' RACE를 통하여 밝혀진 EPSPS의 cDNA는 각각 1176bp의 open reading frame으로 이루어져 있으며 391개의 아미노산을 코딩하고 있었다. 이는 보고된 다른 EPSPS 유전자들과 높은 유사성을 가지고 있다. Genomic southern 결과 들잔디 내에 EPSPS 유전자는 단일copy로 존재하였다. Wild type과 제초제 내성 변이체의 EPSPS 유전자는 6개의 아미노산 시퀀스의 차이를 보였으며 기존에 보고된 EPSPS active site에서 시퀀스의 차이를 나타냈다. 한편 glyphosate의 독성기작의 하나인 EPSPS 효소활성 저해 작용을 알아본 결과, 내성 개체에서 높은(1.5배 이상) EPSPS 활성을 확인할 수 있었다. Northern 분석과 RT-PCR로 제초제 처리 시간에 따른 EPSPS의 발현량을 살펴본 결과, 제초제 처리 후 시간이 경과할수록 발현량이 증가하다가 7일 이후에는 감소하였고, wild type에 비해 glyphosate 내성 선발개체에서 전체적인 발현량이 높음을 알 수 있었다. 본 연구 결과 선발된 glyphosate 내성 돌연변이체는 높은 EPSPS 효소활성 증가 및 EPSPS active site의 아미노산 시퀀스 변화를 통해 원할하고 지속적인 방향족 아미노산의 합성이 가능한 것으로 보여졌다. 본 실험을 통해 얻어진 glyphosate 내성 잔디 돌연변이체와 방법은 앞으로 유용한 제초제 저항성 돌연변이 품종개발 연구에 활용될 수 있을 것으로 기대된다.
본 연구에서는 들잔디와 돌연변이체에서 Glyphosate 내성 관련 유전자인 EPSPS를 코딩하는 cDNA를 분리하여, 시퀸스 비교분석과 발현 양상 차이 등에 관하여 조사하였다. 5'/3' RACE를 통하여 밝혀진 EPSPS의 cDNA는 각각 1176bp의 open reading frame으로 이루어져 있으며 391개의 아미노산을 코딩하고 있었다. 이는 보고된 다른 EPSPS 유전자들과 높은 유사성을 가지고 있다. Genomic southern 결과 들잔디 내에 EPSPS 유전자는 단일copy로 존재하였다. Wild type과 제초제 내성 변이체의 EPSPS 유전자는 6개의 아미노산 시퀀스의 차이를 보였으며 기존에 보고된 EPSPS active site에서 시퀀스의 차이를 나타냈다. 한편 glyphosate의 독성기작의 하나인 EPSPS 효소활성 저해 작용을 알아본 결과, 내성 개체에서 높은(1.5배 이상) EPSPS 활성을 확인할 수 있었다. Northern 분석과 RT-PCR로 제초제 처리 시간에 따른 EPSPS의 발현량을 살펴본 결과, 제초제 처리 후 시간이 경과할수록 발현량이 증가하다가 7일 이후에는 감소하였고, wild type에 비해 glyphosate 내성 선발개체에서 전체적인 발현량이 높음을 알 수 있었다. 본 연구 결과 선발된 glyphosate 내성 돌연변이체는 높은 EPSPS 효소활성 증가 및 EPSPS active site의 아미노산 시퀀스 변화를 통해 원할하고 지속적인 방향족 아미노산의 합성이 가능한 것으로 보여졌다. 본 실험을 통해 얻어진 glyphosate 내성 잔디 돌연변이체와 방법은 앞으로 유용한 제초제 저항성 돌연변이 품종개발 연구에 활용될 수 있을 것으로 기대된다.
This study is the first comprehensive report on the molecular cloning, structural characterization, sequence comparison between wild and mutant types, copy number in the genome, expression features and activities of a gene encoding 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) in Korean lawn g...
This study is the first comprehensive report on the molecular cloning, structural characterization, sequence comparison between wild and mutant types, copy number in the genome, expression features and activities of a gene encoding 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) in Korean lawn grass ($Zoysia$$japonica$). The full length cDNA of the EPSPS from Korean lawn grass ($zj$EPSPS) obtained from a 3' and 5' RACE method was 1540 bp, containing a 1176 bp ORF, a 144 bp leader sequence (5' UTR) and a 220 bp 3' UTR, which was eventually decoded 391 amino acid residues with a molecular mass of 41.74 kDa. The Southern blot detection of the $zj$EPSPS showed that the gene exists as a single copy in the Korean lawn grass genome. Sequence comparison of the $zj$EPSPS gene demonstrated that the glyphosate-tolerant mutant (GT) having a Pro-53 to Ser substitution in the gene seems to have a preferred binding activity of the enzyme to phosphoenol pyruvate(PEP) over glyphosate, which allows the continuous synthesis of aromatic amino acids in the shikimate pathway. From the Northern blotting analysis, the $zj$EPSPS was found to be highly expressed, with continuous increase until 36 hours after 0.5% glyphosate treatment in both wild and mutant samples, but 1.5-fold higher EPSP synthase activity was observed in the tolerant mutant when exposed to the glyphosate treatment. The molecular information of the $zj$EPSPS gene obtained from this study needs to be further dissected to be more effectively applied to the development of gene-specific DNA markers and zoysiagrass cultivars; nevertheless, the glyphosate-tolerant mutant having the featured $zj$EPSPS gene can be provided to turfgrass managers for weed problems with timely adoptable management options.
This study is the first comprehensive report on the molecular cloning, structural characterization, sequence comparison between wild and mutant types, copy number in the genome, expression features and activities of a gene encoding 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) in Korean lawn grass ($Zoysia$$japonica$). The full length cDNA of the EPSPS from Korean lawn grass ($zj$EPSPS) obtained from a 3' and 5' RACE method was 1540 bp, containing a 1176 bp ORF, a 144 bp leader sequence (5' UTR) and a 220 bp 3' UTR, which was eventually decoded 391 amino acid residues with a molecular mass of 41.74 kDa. The Southern blot detection of the $zj$EPSPS showed that the gene exists as a single copy in the Korean lawn grass genome. Sequence comparison of the $zj$EPSPS gene demonstrated that the glyphosate-tolerant mutant (GT) having a Pro-53 to Ser substitution in the gene seems to have a preferred binding activity of the enzyme to phosphoenol pyruvate(PEP) over glyphosate, which allows the continuous synthesis of aromatic amino acids in the shikimate pathway. From the Northern blotting analysis, the $zj$EPSPS was found to be highly expressed, with continuous increase until 36 hours after 0.5% glyphosate treatment in both wild and mutant samples, but 1.5-fold higher EPSP synthase activity was observed in the tolerant mutant when exposed to the glyphosate treatment. The molecular information of the $zj$EPSPS gene obtained from this study needs to be further dissected to be more effectively applied to the development of gene-specific DNA markers and zoysiagrass cultivars; nevertheless, the glyphosate-tolerant mutant having the featured $zj$EPSPS gene can be provided to turfgrass managers for weed problems with timely adoptable management options.
* AI 자동 식별 결과로 적합하지 않은 문장이 있을 수 있으니, 이용에 유의하시기 바랍니다.
문제 정의
Compared with the wild type, the selected tolerant mutant to glyphosate was observed to delay some physiological disorder, allowing them visually more active for long periods and providing turfgrass managers under weed problems with timely adoptable management options. This was the first report on the cloning of EPSPS in the Korean lawn grass and the tolerant mechanisms explained by the amino acid substitution and the expression difference in the gene which was derived from the gamma-ray mutagenesis. The gene information obtained from this study will be further dissected to be more effectively applied to the development of gene-specific DNA markers and tolerant turfgrass cultivars.
제안 방법
plasmid DNA purification Kit (iNtRON Biotechnology, Seoul, Korea) according to the manufacturer’s instructions. A sequencing of the isolated DNA was performed by using the BigDyeⓇ Terminator v1.1 Cycle Sequencing Kit (Applied Biosystems, Foster City, U.S.A) and analyzed with the Applied Biosystems 3130xl Genetic Analyzer (Applied Biosystems, Foster City, U.S.A) in the Advanced Radiation Technology Institute of Korea Atomic Energy Research Institute in Jeongeup, Korea. The PCR was performed with 25 cycles at 96℃ for 10 sec, 50℃ for 5 sec, 72℃ for 4 min using a gradient thermal cycler (TP600, TaKaRa, Shiga, Japan).
, 2008b). One of three tolerance lines was used to clone and characterize the EPSPS gene in this study.
0 (Takara, Japan) with a template of 1 µg of total RNA and a degenerated primer. To obtain the EPSPS gene sequence, a partial cDNA was amplified with a degenerated primer, which was designed from the conserved DNA sequences of Lolium rigidum (AF349754) and Eleusine indica (AJ417033) by using a ClustalW program (http://www.ebi.ac.uk). The sense/antisense primers were 5'-TGTTGATTGTTTCCTT GGCACTG-3' (EPSPS-F) and 5'-ACATCTCTGATAGCAGTTGG-3' (EPSPS-R).
대상 데이터
All the inserted sequences were checked for their homology using the BLAST program in NCBI sequence database and another program in the GenBank/EMBL/DDBJ sequence database. The open reading frame (ORF) and conserved domain were generated by the NCBI BLASTN program (http://www.ncbi.nlm.nih.gov/). Sequence alignment, ORF translation, molecular weight calculation of the predicted proteins and a structural analysis of the deduced proteins were carried out by a program on the website (http://kr.
이론/모형
The EPSPS activity was measured by the production of inorganic phosphate using the malachite green dye assay by Lanzetta et al. (1979). The standard reaction was carried out at 30℃ in a final volume of a 100 μL mixture (pH 7.
Structural comparison between the wild type (A) and mutant line (B). The three-dimensional structures of EPSPS proteins were predicted using the automated SWISS-MODEL program. The indicated arrows represent the conformation changes in the EPSPS due to the Pro to Ser substitution in the codon region.
성능/효과
However, the gene was not cloned from Korean lawn grass. In this study, the gene encoding Korean lawn grass EPSPS was isolated and the EPSPS gene sequence of some tolerant mutant lines was compared with that of a wild type. The transcription and sequence information obtained in this study might be further applied to other studies including the development of DNA markers and resistant cultivars to the glyphosate in the Korean lawn grass.
1B). The deduced amino acid sequence of zjEPSPS show a 98% identity to eiEPSPS from Eleusine indica (CAD01095), 95% osEPSPS from Oryza sative (BAA32276), 92% zmEPSPS (CAA44974), 87% lrEPSPS (AAK20397). From the phylogenetic analysis using most of the published EPSPS genes, zjEPSPS was tightly sub-grouped with eiEPSPS, then with other EPSPS to form a highly homologous large group (data not shown).
The five clones were sequenced and blasted using the NCBI BLASTN program. The sequence comparison revealed that the sequences of the five clones are highly similar to the reported EPSPS sequences, ranging from 75 to 93% with higher homology of 93% with Eleusine indica (AJ417033) and 91% with Zea mays (X63374) (Table 1).
3). Thirteen single-nucleotide differences were identified between the wild type and the mutant, which were consistent among all the cDNAs analyzed. Seven of the detected nucleotide changes, G84 (wild type) to A (GT), C159 (wild type) to T (GT), T402 (wild type) to C (GT), C465 (wild type) to G (GT), C774 (wild type) to G (GT), G834 (wild type) to A (GT), and G924 (wild type) to A (GT), correspond to silent changes in the codon wobble positions for Gly-28, Pro-53, Met-134, Pro-155, Pro-258, Asp-278, and Glu-308, respectively.
후속연구
This was the first report on the cloning of EPSPS in the Korean lawn grass and the tolerant mechanisms explained by the amino acid substitution and the expression difference in the gene which was derived from the gamma-ray mutagenesis. The gene information obtained from this study will be further dissected to be more effectively applied to the development of gene-specific DNA markers and tolerant turfgrass cultivars.
참고문헌 (36)
Abraham, Z., M. Martinez, P. Carbonero, and I. Diaz. 2006. Structural and functional diversity within the cystatin gene family of Hordeum vulgare. J. Exp. Bot. 57:4245-4255.
Baerson, S.R., D.J. Rodriguez, M. Tran, Y. Feng, N.A. Biest, and G.M. Dill. 2002b. Glyphosate-resistant goose grass. Identification of a mutation in the target enzyme 5-enolpyruvylshikimate- 3-phosphate synthase. Plant Physiol. 129:1265-1275.
Charles, I.G., J.W. Keyte, W.J. Brammar, M. Smith, and A.R. Hawkins. 1986. The isolation and nucleotide sequence of the complex AROM locus of Aspergillus nidulans. Nucleic Acids Res. 14:2201-2213.
Choi, J.S., B.J. Ahn, and G.M. Yang. 1997. Distribution of native zoysia grasses (Zoysia spp.) in the south and west coastal regions of Korea and classification using morphological characteristics. J. Kor. Soc. Hort. Sci. 38:327-332.
Comai, L.D., D. Facciotti, W.R. Hiatt, G. Thompson, R.E. Rose, and D.M. Stalker. 1985. Expression in plants of a mutant aroA gene from Salmonella tymphimurium confers tolerance to glyphosate. Nature 317:741-744.
Duncan, K., A. Lewendon, and J.R. Coggins. 1984. The complete amino acid sequence of Escherichia coli 5-enolpyruvylshikimate- 3-phosphate synthase. FEBS Lett. 170:59-63.
Duncan, K., R.M. Edwards, and J.R. Coggins. 1987. The pentafunctional arom enzyme of Saccharomyces cerevisiae is a mosaic of mono- functional domains. Biochem. J. 246:375-386.
Doyle, J.J. and J.L. Doyle. 1987. A rapid DNA isolation procedure for small quantities of fresh leaf tissue. Phytochemical Bulletin. 19:11-15.
Gasser, C.S., J.A. Winter, C.M. Hironaka, and D.M. Shah. 1988. Structure, expression, and evolution of the 5-enolpyruvylshikimate- 3-phosphate synthase genes of petunia and tomato. J. Biol. Chem. 263:4280-4287.
Hong, J.K., J.E. Hwang, T.-H. Park, Y.-X. Zang, S.C. Lee, S.-J. Kwon, J.-H. Mun, H.U. Kim, J.A. Kim, M. Jin, J.S. Kim, S.I. Lee, M.-H. Lim, Y.K. Hur, C.O. Lim, and B.-S. Park. 2008. Identification and characterization of the phytocystatin family from Brassica rapa. J. Plant Biotech. 4:317-327.
Jin, D., W. Lu, S. Ping, W. Zhang, J. Chen, B. Dun, R. Ma, Z. Zhao, J. Sha, L. Li, Z. Yang, M. Chen, and M. Lin. 2007. Identification of a new gene encoding EPSPS with high glyphosate resistance from the metagenomic library. Curr. Microbiol. 55:350-355.
Klee, H.J., Y.M. Muskopf, and C.S. Gasser. 1987. Cloning of and Arabidopsis thaliana gene encoding 5-enolpyruvylshikimate- 3-phosphate synthase: sequence analysis and manipulation to obtain glyphosate-tolerant plants. Mol. Gen. Genet. 210:437-442.
Lee, H.J., G.-J. Lee, D.S. Kim, J.B. Kim, J.H. Ku, and S.-Y. Kang. 2008a. Determination of the optimum dose range for a mutation induction of turfgrasses by a gamma-ray. Kor. Turfgrass Sci. 22:25-34.
Lee, H.J., G.-J. Lee, D.S. Kim, J.B. Kim, J.H. Ku, and S.-Y. Kang. 2008b. Selection and physiological characterization of glyphosate-resistant zoysia grass mutants derived from a gamma ray irradiation. Kor. J. Hort. Sci. Technol. 26:454-463.
Marchler-Bauer, A. and S.H. Bryant. 2004. CD-Search: protein domain annotations on the fly. Nucleic Acids Res. 32:2327-2331.
Madera, M., C. Vogel, S.K. Kummerfeld, C. Chothia, and J. Gough. 2004. The SUPERFAMILY database in 2004: additions and improvements. Nucl. Acids Res. 32:235-239.
McDowell, L.M., A. Schmidt, E.R. Cohen, D.R. Studelska, and J. Schaefer. 1996. Structural constraints on the ternary complex of 5-enolpyruvylshikimate-3-phosphate synthase from rotationalecho double-resonance NMR. J. Mol. Biol. 256:160-171.
Oliveira, J.S., C.A. Pinto, L.A. Basso, and D.S. Santos. 2001. Cloning and overexpression in soluble form of functional shikimate kinase and 5-enolpyruvylshikimate-3-phosphate synthase enzymes from Mycobacterium tuberculosis. Protein Expr. Purif. 22:430-435.
Padgete, S.R., D.B. Re, C.S. Gasser, D.A. Eichholtz, R.B. Frazier, C.M. Hironaka, E.B. Levine, D.M. Shah, R.T. Fraley, and G.M. Kishore. 1991. Site-directed mutagenesis of a conserved region of the 5-enolpyruvyl shikimate-3-phosphate synthase active site. J. Biol. Chem. 266:22364-22369.
Priestman, M.A., M.L. Healy, T. Funke, A. Becker, and E. Schonbrunn. 2005. Molecular basis for the glyphosateinsensitivity of the reaction of 5-enolpyruvylshikimate-3- phosphate synthase with shikimate. FEBS Letter 579:5773-5780.
Schonbrunn, E., S. Eschenburg, W.A. Shuttleworth, J.V. Schloss, N. Amrhein, J.N. Evans, and W. Kabsch. 2001. Interaction of the herbicide glyphosate with its target enzyme 5- enolpyruvylshikimate 3-phosphate synthase in atomic detail. Proc. Natl. Acad. Sci. USA 98:1376-1380.
Stalker, D.M., W.R. Hiatt, and L. Comai. 1985. A single amino acid substitution in the enzyme 5-enolpyruvyl shikimate-3-phosphate synthase confers resistance to the herbicide glyphosate. J. Biol. Chem. 260:4724-4728.
Steinrucken, H.C. and N. Amrhein. 1980. The herbicide glyphosate is a potent inhibitor of 5-enolpyruvyl shikimate-3-phosphate synthase. Biochem. Biophys. Res. Commun. 94:1207-1212.
Toyama, K., C.H. Bae, J.G. Kang, Y.P. Lim, T. Adachi, K.Z. Riu, P.S. Song, and H.Y. Lee. 2003. Production of herbicidetolerant zoysiagrass by Agrobacterium-mediated transformation. Mol. Cells 16:269-272.
Wang, Y.X., J.D. Jones, S.C. Weller, and P.B. Goldsbrough. 1991. Expression and stability of amplified genes encoding 5-enolpyruvylshikimate-3-phosphate synthase in glyphosatetolerant tobacco cells. Plant Mol. Biol. 17:1127-1138.
Yi, Y.D. Qiao, L. Bai, H. Xu, Y. Li, X. Wang, and Y. Cao. 2007. Cloning, expression, and functional characterization of the Dunaliella salina 5-enolpyruvylshikimate-3-phosphate synthase gene in Escherichia coli. J. Microbiol. 45:153-157.
Yu, Q., A. Cairns, and S. Powles. 2007. Glyphosate, paraquat and ACCase multiple herbicide resistance evolved in a Lolium rigidum biotype. Planta 225:499-513.
※ AI-Helper는 부적절한 답변을 할 수 있습니다.