Name: a simple method for isolation of soybean protoplasts and application to transient gene expression analyses
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이 작품은 국립 과학 재단 (NSF-PGRP-IOS-1339388)에서 식물 게놈 연구 프로그램에 의해 지원 되었다.

1입니다. 식물의 성장
<ol><li>콩에 대 한 사용자 지정 토양 혼합에 25 ° C에서 긴 하루 조건 (16 h 1500 µmol m-2의 -1에 빛)에서 온실에서 13cm 냄비에 5-10 콩 씨앗 (윌리엄스 82)를 뿌리 다 (1: 토양, 진주 암 및 어 뢰 모래의 비율이 1:1).</li>
</ol>2입니다. 플라스 미드 DNA의 준비
<ol><li>살 균 피 펫 팁 또는 이쑤시개 사용 하, 단일 식민지 또는 냉동된 글리세롤 재고의 대장...

<ol>
	<li>Melchers, G., Labib, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Somatic+hybridisation+of+plants+by+fusion+of+protoplasts.">Somatic hybridisation of plants by fusion of protoplasts.</a> Mol. Gen. Genet. 135 (4), 277-294 (1974).</li><li>Gresshoff, P. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+culture+of+white+clover:+callus,+suspension,+protoplast+culture,+and+plant+regeneration.">In vitro culture of white clover: callus, suspension, protoplast culture, and plant regeneration.</a> Bot. Gaz. 141 (2), 157-164 (1980).</li><li>L&#246;rz, H., Baker, B., Schell, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+transfer+to+cereal+cells+mediated+by+protoplast+transformation.">Gene transfer to cereal cells mediated by protoplast transformation.</a> Mol. Gen. Genet. 199 (2), 178-182 (1985).</li><li>Hayashimoto, A., Li, Z., Murai, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+polyethylene+glycol-mediated+protoplast+transformation+system+for+production+of+fertile+transgenic+rice+plants.">A polyethylene glycol-mediated protoplast transformation system for production of fertile transgenic rice plants.</a> Plant Physiol. 93 (3), 857-863 (1990).</li><li>Koop, H. -. U., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Integration+of+foreign+sequences+into+the+tobacco+plastome+via+polyethylene+glycol-mediated+protoplast+transformation.">Integration of foreign sequences into the tobacco plastome via polyethylene glycol-mediated protoplast transformation.</a> Planta. 199 (2), 193-201 (1996).</li><li>Hrazdina, G., Wagner, G. J., Siegelman, H. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Subcellular+localization+of+enzymes+of+anthocyanin+biosynthesis+in+protoplasts.">Subcellular localization of enzymes of anthocyanin biosynthesis in protoplasts.</a> Phytochemistry. 17 (1), 53-56 (1978).</li><li>Lin, W., Wittenbach, V. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Subcellular+localization+of+proteases+in+wheat+and+corn+mesophyll+protoplasts.">Subcellular localization of proteases in wheat and corn mesophyll protoplasts.</a> Plant Physiol. 67 (5), 969-972 (1981).</li><li>V&#246;geli-Lange, R., Wagner, G. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Subcellular+localization+of+cadmium+and+cadmium-binding+peptides+in+tobacco+leaves.">Subcellular localization of cadmium and cadmium-binding peptides in tobacco leaves.</a> Plant Physiol. 92 (4), 1086-1093 (1990).</li><li>Chen, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+highly+efficient+transient+protoplast+system+for+analyzing+defence+gene+expression+and+protein-protein+interactions+in+rice.">A highly efficient transient protoplast system for analyzing defence gene expression and protein-protein interactions in rice.</a> Mol. Plant Pathol. 7 (5), 417-427 (2006).</li><li>Wu, F. -. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tape-Arabidopsis+Sandwich-a+simpler+Arabidopsis+protoplast+isolation+method.">Tape-Arabidopsis Sandwich-a simpler Arabidopsis protoplast isolation method.</a> Plant methods. 5 (1), 16 (2009).</li><li>Christensen, A. H., Sharrock, R. A., Quail, P. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Maize+polyubiquitin+genes:+structure,+thermal+perturbation+of+expression+and+transcript+splicing,+and+promoter+activity+following+transfer+to+protoplasts+by+electroporation.">Maize polyubiquitin genes: structure, thermal perturbation of expression and transcript splicing, and promoter activity following transfer to protoplasts by electroporation.</a> Plant Mol. Biol. 18 (4), 675-689 (1992).</li><li>Marcotte, W. R., Bayley, C. C., Quatrano, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+a+wheat+promoter+by+abscisic+acid+in+rice+protoplasts.">Regulation of a wheat promoter by abscisic acid in rice protoplasts.</a> Nature. 335 (6189), 454-457 (1988).</li><li>Dron, M., Clouse, S. D., Dixon, R. A., Lawton, M. A., Lamb, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glutathione+and+fungal+elicitor+regulation+of+a+plant+defense+gene+promoter+in+electroporated+protoplasts.">Glutathione and fungal elicitor regulation of a plant defense gene promoter in electroporated protoplasts.</a> P. Natl. A. Sci. USA. 85 (18), 6738-6742 (1988).</li><li>Cocking, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+method+for+the+isolation+of+plant+protoplasts+and+vacuoles.">A method for the isolation of plant protoplasts and vacuoles.</a> Nature. 187 (4741), 962-963 (1960).</li><li>Zhang, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transgenic+rice+plants+produced+by+electroporation-mediated+plasmid+uptake+into+protoplasts.">Transgenic rice plants produced by electroporation-mediated plasmid uptake into protoplasts.</a> Plant Cell Rep. 7 (6), 379-384 (1988).</li><li>Fromm, M., Taylor, L. P., Walbot, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+of+genes+transferred+into+monocot+and+dicot+plant+cells+by+electroporation.">Expression of genes transferred into monocot and dicot plant cells by electroporation.</a> P. Natl. A. Sci. USA. 82 (17), 5824-5828 (1985).</li><li>Crossway, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Integration+of+foreign+DNA+following+microinjection+of+tobacco+mesophyll+protoplasts.">Integration of foreign DNA following microinjection of tobacco mesophyll protoplasts.</a> Mol. Gen. Genet. 202 (2), 179-185 (1986).</li><li>Holm, P. B., Olsen, O., Schnorf, M., Brinch-Pedersen, H., Knudsen, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transformation+of+barley+by+microinjection+into+isolated+zygote+protoplasts.">Transformation of barley by microinjection into isolated zygote protoplasts.</a> Transgenic Res. 9 (1), 21-32 (2000).</li><li>Schapire, A. L., Lois, L. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simplified+and+rapid+method+for+the+isolation+and+transfection+of+Arabidopsis+leaf+mesophyll+protoplasts+for+large-scale+applications.">A simplified and rapid method for the isolation and transfection of Arabidopsis leaf mesophyll protoplasts for large-scale applications.</a> Plant Signal Transduction: Methods and Protocols. 1363, 79-88 (2016).</li><li>Niedz, R. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regeneration+of+somatic+embryos+from+sweet+orange+(C.+sinensis)+protoplasts+using+semi-permeable+membranes.">Regeneration of somatic embryos from sweet orange (C. sinensis) protoplasts using semi-permeable membranes.</a> Plant Cell Tiss. Org. Cult. 84 (3), 353-357 (2006).</li><li>Sheng, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protoplast+isolation+and+plant+regeneration+of+different+doubled+haploid+lines+of+cauliflower+(Brassica+oleracea+var.+botrytis).">Protoplast isolation and plant regeneration of different doubled haploid lines of cauliflower (Brassica oleracea var. botrytis).</a> Plant Cell Tiss. Org. 107 (3), 513-520 (2011).</li><li>Grun, P., Chu, L. -. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+plants+from+protoplasts+of+Solanum+(Solanaceae).">Development of plants from protoplasts of Solanum (Solanaceae).</a> Am. J. Bot. 65 (5), 538-543 (1978).</li><li>Davey, M. R., Anthony, P., Power, J. B., Lowe, K. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plant+protoplasts:+status+and+biotechnological+perspectives.">Plant protoplasts: status and biotechnological perspectives.</a> Biotechnol. Adv. 23 (2), 131-171 (2005).</li><li>Pitzschke, A., Persak, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Poinsettia+protoplasts-a+simple,+robust+and+efficient+system+for+transient+gene+expression+studies.">Poinsettia protoplasts-a simple, robust and efficient system for transient gene expression studies.</a> Plant methods. 8 (1), 14 (2012).</li><li>Yoo, S. -. D., Cho, Y. -. H., Sheen, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Arabidopsis+mesophyll+protoplasts:+a+versatile+cell+system+for+transient+gene+expression+analysis.">Arabidopsis mesophyll protoplasts: a versatile cell system for transient gene expression analysis.</a> Nat. Protoc. 2 (7), 1565 (2007).</li><li>Sedivy, E. J., Wu, F., Hanzawa, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Soybean+domestication:+the+origin,+genetic+architecture+and+molecular+bases.">Soybean domestication: the origin, genetic architecture and molecular bases.</a> New Phytol. 214 (2), 539-553 (2017).</li><li>Yang, Y., Li, R., Qi, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+analysis+of+plant+promoters+and+transcription+factors+by+agroinfiltration+of+tobacco+leaves.">In vivo analysis of plant promoters and transcription factors by agroinfiltration of tobacco leaves.</a> Plant J. 22 (6), 543-551 (2000).</li><li>Marion, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Systematic+analysis+of+protein+subcellular+localization+and+interaction+using+high-throughput+transient+transformation+of+Arabidopsis+seedlings.">Systematic analysis of protein subcellular localization and interaction using high-throughput transient transformation of Arabidopsis seedlings.</a> Plant J. 56 (1), 169-179 (2008).</li><li>Wu, H. -. Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=AGROBEST:+an+efficient+Agrobacterium-mediated+transient+expression+method+for+versatile+gene+function+analyses+in+Arabidopsis+seedlings.">AGROBEST: an efficient Agrobacterium-mediated transient expression method for versatile gene function analyses in Arabidopsis seedlings.</a> Plant methods. 10 (1), 19 (2014).</li><li>Govindarajulu, M., Elmore, J. M., Fester, T., Taylor, C. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+constitutive+viral+promoters+in+transgenic+soybean+roots+and+nodules.">Evaluation of constitutive viral promoters in transgenic soybean roots and nodules.</a> Mol Plant Pathol. 21 (8), 1027-1035 (2008).</li><li>Nagamatsu, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+analysis+of+soybean+genes+involved+in+flavonoid+biosynthesis+by+virus-induced+gene+silencing.">Functional analysis of soybean genes involved in flavonoid biosynthesis by virus-induced gene silencing.</a> Plant Biotechnol. J. 5 (6), 778-790 (2007).</li><li>Juvale, P. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Temporal+and+spatial+Bean+pod+mottle+virus-induced+gene+silencing+in+soybean.">Temporal and spatial Bean pod mottle virus-induced gene silencing in soybean.</a> Mol. Plant Pathol. 13 (9), 1140-1148 (2012).</li><li>Zhang, C., Bradshaw, J. D., Whitham, S. A., Hill, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+development+of+an+efficient+multipurpose+bean+pod+mottle+virus+viral+vector+set+for+foreign+gene+expression+and+RNA+silencing.">The development of an efficient multipurpose bean pod mottle virus viral vector set for foreign gene expression and RNA silencing.</a> Plant Physiol. 153 (1), 52-65 (2010).</li><li>Lin, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+mesophyll+protoplasts+from+mature+leaves+of+soybeans.">Isolation of mesophyll protoplasts from mature leaves of soybeans.</a> Plant Physiol. 73 (4), 1067-1069 (1983).</li><li>Yi, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+single-repeat+MYB+transcription+factor,+GmMYB176,+regulates+CHS8+gene+expression+and+affects+isoflavonoid+biosynthesis+in+soybean.">A single-repeat MYB transcription factor, GmMYB176, regulates CHS8 gene expression and affects isoflavonoid biosynthesis in soybean.</a> Plant J. 62 (6), 1019-1034 (2010).</li><li>Faria, J. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+NAC+domain-containing+protein,+GmNAC6,+is+a+downstream+component+of+the+ER+stress-and+osmotic+stress-induced+NRP-mediated+cell-death+signaling+pathway.">The NAC domain-containing protein, GmNAC6, is a downstream component of the ER stress-and osmotic stress-induced NRP-mediated cell-death signaling pathway.</a> BMC Plant Biol. 11 (1), 129 (2011).</li><li>Kidokoro, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Soybean+DREB1/CBF-type+transcription+factors+function+in+heat+and+drought+as+well+as+cold+stress-responsive+gene+expression.">Soybean DREB1/CBF-type transcription factors function in heat and drought as well as cold stress-responsive gene expression.</a> Plant J. 81 (3), 505-518 (2015).</li><li>Sun, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeted+mutagenesis+in+soybean+using+the+CRISPR-Cas9+system.">Targeted mutagenesis in soybean using the CRISPR-Cas9 system.</a> Sci Rep-UK. 5, 10342 (2015).</li><li>Karimi, M., Inz&#233;, D., Depicker, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=GATEWAY&#8482;+vectors+for+Agrobacterium-mediated+plant+transformation.">GATEWAY&#8482; vectors for Agrobacterium-mediated plant transformation.</a> Trends Plant Sci. 7 (5), 193-195 (2002).</li><li>Xia, Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Positional+cloning+and+characterization+reveal+the+molecular+basis+for+soybean+maturity+locus+E1+that+regulates+photoperiodic+flowering.">Positional cloning and characterization reveal the molecular basis for soybean maturity locus E1 that regulates photoperiodic flowering.</a> P. Natl. A. Sci. USA. 109 (32), E2155-E2164 (2012).</li></ol>

콩 protoplasts 및 과도 식 연구에 응용 프로그램의 격리에 대 한이 정서의 철저 하 게 테스트 하 고 우리의 실험실에서 아주 잘 작동. 절차는 간단 하 고 쉽게 고 일반 장비 및 최소 비용 요구. 우리의 프로토콜 이전에 보고 된 방법34,35,36,,3738에 비해 균일 한 고품질 protoplasts의 ?...

Protoplasts는 식물 세포는 세포 벽 제거. 그들은 대부분의 기능을 유지 하 고 식물 세포의 활동, protoplasts는 관찰 하 고 평가 하는 다양 한 세포 이벤트, 좋은 모델 시스템 하 고 체세포 혼성1 을 공부 하 고 중생2공장에 유용한 도구. Protoplasts 공장 변환3,,45, 세포 벽 세포로 DNA의 통로 차단 그렇지 않으면 이후 널리 이용 되어 있다. Protoplasts 생리 적인 응답의 일부 및 따라서 단백질 subcellular 지 방화6,7,8, 공부 하는 기초 연구에 근본적인 가치를 제공 하 고 그대로 식물의 세포 프로세스 보유 단백질 단백질 상호 작용9,10, 발기인 활동11,,1213 에 셀 살고 있다.
식물 protoplasts의 196014 에 처음 알려졌다 고 절연 및 protoplasts의 변환에 대 한 프로토콜 개발 및 최적화 되었습니다. 원생 동물 격리의 표준 절차는 잎의 절단 및 세포 벽, 비 소화 조직 파편에서 출시 된 protoplasts의 분리에 의해 다음의 효소 소화를 포함 한다. 전환 전략 electroporation15,16, microinjection17,18및 폴 리 에틸렌 글리콜 기반 (PEG)4,,519 방법을 포함 합니다. 다양 한 종의 원생 동물 격리, 감귤 류20, 브라시카21, Solanaceae22 및 다른 장식적인 식물 가족23,24를 포함 하 여 성공 보고 되었습니다. 다양 한 조직 유형에 다양 한 종에 사용 하 고, 표현의 과도 애기 mesophyll 원생 동물 (TEAMP)에서 애기 thaliana 모델 식물의 잎에서 분리 된 시스템 잘 설립된25 되었습니다. 그리고 다양 한 응용 프로그램을 광범위 하 게 채택.
콩 (최대 글리신 (L.) Merr.) 가장 중요 한 단백질의 하나 이며 기름 작물26. 애기 와 달리 쌀, 유전자 변형 콩 식물을 얻기 보다 어렵고 낮은 효율 될 알려져 있다. Agrobacterium tumefaciens-중재 침투는 널리 사용 되 고 담배27 에서 상피 세포와 애기28,29, 모 종 변이 유전자 표현 연구 반면 Agrobacterium rhizogenes 콩30털 뿌리의 변화에 대 한 사용 되었습니다. 바이러스 유도 유전자 입을 접근 downregulation 대상 유전자31,32 와 과도 식33 의 체계적인 방식으로 활용 되어 있다. Protoplasts 이러한 접근에 대 한 소중 하 고 다양 한 대안을 제공합니다. Protoplasts 콩의 지상 자료에서 얻어질 수 있다 고 신속 하 고 동기화 transgene 식. 그러나, 198334에 콩 protoplasts의 초기 성공 격리, 이후 제한 보고서 콩35,,3637, protoplasts의 응용 프로그램에 38, 콩 protoplasts의 상대적으로 낮은 수확량 때문에 주로.
여기, 우리 콩 protoplasts의 격리에 대 한 간단 하 고 효율적인 프로토콜 및 변이 유전자 표현 연구에 대 한 응용 프로그램을 설명합니다. 콩 모 종에서 젊은 unifoliate 잎을 사용 하 여, 몇 시간 안에 많은 양의 중요 한 protoplasts 얻을 수 있었습니다. 또한, 우리는 간단 하 고 저비용 고효율 콩 protoplasts에 DNA를 전달 하는 말뚝 칼슘 중재 하는 변환 메서드를 최적화 했습니다.

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			<td height="21" style="height:21px;width:256px;">MES</td>
			<td style="width:181px;">Sigma Aldrich&#160;</td>
			<td style="width:119px;">M8250-100G</td>
			<td style="width:167px;"></td>
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			<td height="21" style="height:21px;">Cellulase CELF</td>
			<td>Worthington Biological Corporation</td>
			<td>LS002611</td>
			<td></td>
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			<td height="21" style="height:21px;">Pectolyase Y-23</td>
			<td>BioWorld</td>
			<td>9033-35-6</td>
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			<td height="21" style="height:21px;">CELLULASE &#34;ONOZUKA&#34; R-10</td>
			<td>yakult</td>
			<td>10g</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;">MACEROZYME R-10</td>
			<td>yakult</td>
			<td>10g</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;">Mannitol</td>
			<td>ICN Biomedicals&#160;</td>
			<td>152540</td>
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			<td height="21" style="height:21px;">CaCl2</td>
			<td>Fisher&#160;</td>
			<td>C79-500g&#160;</td>
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		<tr height="21">
			<td height="21" style="height:21px;">BSA</td>
			<td>NEB</td>
			<td>R3535S</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;">DTT</td>
			<td>Sigma Aldrich&#160;</td>
			<td>D5545-5G</td>
			<td></td>
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			<td height="21" style="height:21px;">NaCl</td>
			<td>Sigma Aldrich&#160;</td>
			<td>S7653-1kg</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;">KCl</td>
			<td>Fisher&#160;</td>
			<td>P217-500g&#160;</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;">MgCl2</td>
			<td>Sigma Aldrich&#160;</td>
			<td>M8266-100g</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;">PEG4000</td>
			<td>Fluka</td>
			<td>81240</td>
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		<tr height="21">
			<td height="21" style="height:21px;">nylon mesh</td>
			<td>carolina</td>
			<td>652222N</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:256px;">Tissue Culture Plates&#160;</td>
			<td>USA Scientific</td>
			<td>CC7682-7506</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:256px;">Razor Blades</td>
			<td style="width:181px;">Fisher</td>
			<td>12-640</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">hemacytometer</td>
			<td>hausserscientific</td>
			<td>1483</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:256px;">QIAprep Spin Miniprep Kit</td>
			<td style="width:181px;">Qiagen</td>
			<td>27104</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:256px;">EZNA plasmid miniprep kit</td>
			<td style="width:181px;">Omega</td>
			<td>D6942-01</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:256px;">GeneJET Plasmid Miniprep Kit</td>
			<td style="width:181px;">Thermo Scientific</td>
			<td>K0502</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:256px;">Centrifuge 5810</td>
			<td style="width:181px;">eppendorf</td>
			<td style="width:119px;">5811000827</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:256px;">Centrifuge 5424</td>
			<td style="width:181px;">eppendorf</td>
			<td>22620401</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:256px;">Jencons Powerpette Plus Pipet Controller</td>
			<td style="width:181px;">Jencons</td>
			<td>14526-202</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:25px;width:256px;">Zeiss 710 Confocal Microscope</td>
			<td style="width:181px;">Zeiss</td>
			<td style="width:119px;">N/A</td>
			<td></td>
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			<td height="33" style="height:33px;width:256px;">Nonstick, RNase-free Microfuge Tubes, 1.5 mL</td>
			<td style="width:181px;">Ambion</td>
			<td style="width:119px;">AM12450</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:256px;">15 mL Centrifuge Tubes</td>
			<td style="width:181px;">Denville</td>
			<td style="width:119px;">C1018-P</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:256px;">50 mL Centrifuge Tubes</td>
			<td style="width:181px;">Denville</td>
			<td style="width:119px;">C1060-P</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:256px;">Newborn Calf Serum</td>
			<td style="width:181px;">Thermo Scientific</td>
			<td style="width:119px;">16010159</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:256px;">Soil</td>
			<td style="width:181px;">Ingram&#39;s Nursery</td>
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			<td height="21" style="height:21px;width:256px;">perlite</td>
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			<td height="21" style="height:21px;width:256px;">Torpedo Sand</td>
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			<td height="21" style="height:21px;width:256px;">LB Broth, Lennox (Powder)</td>
			<td style="width:181px;">Fisher</td>
			<td style="width:119px;">BP1427-500</td>
			<td></td>
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</table>

a simple method for isolation of soybean protoplasts and application to transient gene expression analyses

콩 (최대 글리신 (L.) Merr.)는 중요 한 작물 종 이며 유전 그리고 생 화 확 적인 통로의 연구에 대 한 콩과 모델 되고있다. 따라서, 그것은 콩에서 효율적인 변이 유전자 표현 시스템을 설정 하는 것이 중요입니다. 여기, 우리 콩 protoplasts의 준비에 대 한 간단한 프로토콜 및 일시적 기능 분석에 대 한 응용 프로그램을 보고합니다. 우리는 젊은 unifoliate 잎 콩 모 종에서 높은 품질 protoplasts의 대량에서 결과 발견. 못 칼슘 중재 하는 변환 메서드를 최적화 하 여 우리 콩 unifoliate protoplasts를 사용 하 여 높은 변환 효율을 달성. 이 시스템 라이브 콩 셀에 복잡 한 규제 및 신호 메커니즘의 검사에 대 한 효율적이 고 다재 다능 한 모델을 제공 하 고 더 나은 하는 데 도움이 콩의 다양 한 세포, 발달 및 생리 적 프로세스를 이해할 수 있습니다.

다른 장기 10 일 오래 된 콩의 원생 동물 준비 (그림 1)에 대 한 테스트 및 수율 (그림 2) 현미경 관찰 했다. Hypocotyl 및 epicotyl에서 세포 벽 거의 소화 했다, 하 고 일부 셀 (그림 2B, 2 C) 서로 게 연결 된. 떡 (그림 2D)와 루트 (그림 2A), 세포 벽은 세포의 작...

Watch this Scientific Journal Video about A Simple Method for Isolation of Soybean Protoplasts and Application to Transient Gene Expression Analyses at JoVE.com

A Simple Method for Isolation of Soybean Protoplasts and Application to Transient Gene Expression Analyses

우리는 라이브 셀에 복잡 한 규제 및 신호 메커니즘을 연구를 많은 양의 콩 protoplasts의 준비를 위한 간단 하 고 효율적인 프로토콜을 개발.

콩 Protoplasts 및 변이 유전자 표정 분석 응용 프로그램의 격리에 대 한 간단한 방법

Soybean (Glycine max (L.) Merr.) is an important crop species and has become a legume model for the studies of genetic and biochemical pathways. ...

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