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 대장균 의 유전자를 포함 하는 플라스 미드를 운반 하 고 20 mL 50 mL 플라스 크에 적절 한 항생제와 Luria Bertani (파운드) 액체 매체에 접종.</li>
	<li>통에 하룻밤 37 ° C에서 플라스 크를 품 어. 5 분 동안 실 온에서 12000 x g에서 정지 centrifuging와 삭제는 상쾌한 박테리아를 수집 합니다. 추출 하 고 플라스 미드 플라스 미드 준비 장비의 제조 업체의 절차에 따라 정화.</li>
</ol>3. 원생 동물 격리
<ol><li>10 일 된 콩 모 종 (그림 1)에서 새로 확장 된 unifoliate 나뭇잎을 잘라. 참고: 적절 한 발달 단계에서 잎의 선택 콩 원생 동물 준비에 대 한 성공에 열쇠 이다. 만 그냥 확장된 잎을 사용 하 여 초기 발달 단계에서. 성숙한 잎, 세포 벽 소화 하기 어렵게 된다.</li>
	<li>신선한 면도날으로는 midrib unifoliate 잎 및 0.5-1에 남아 m m 스트립 다음 컷에서 제거 합니다. 참고: 10 mL 효소 솔루션에 소화 두 unifoliate 잎 10 개 이상의 변환에 대 한 충분 한 protoplasts를 줄 것 이다.</li>
	<li>집게, 전송의 쌍을 사용 하 여 리프 스트립 즉시 고 부드럽게 15 mL 튜브에 갓된 효소 솔루션 (표 1) 10 mL에. 진공 침투 잎은 실 온에서 15 분 동안 스트립.</li>
	<li>부드러운 실 온에서 4-6 h에 대 한 낮은 조명에서 동요 (40 rpm)와 효소 솔루션에서 리프 스트립을 품 어. 효소 솔루션 변하기 노란색-녹색 protoplasts 해제 됩니다 확인 하십시오. 현미경 (X10) 효소/protoplasts 솔루션을 확인 하십시오. 참고: 릴리스 protoplasts 모양, 소화 되지 않은 세포는 불규칙 한 또는 타원형 모양을 하는 동안 구형은.</li>
	<li>부드럽게 붓는 의해 50 mL 튜브에 효소/원생 동물 솔루션의 10 mL를 전송 하 고 소화를 막으려고 실 온에서 W5 솔루션 (표 1) 10 mL를 추가 합니다. 부드럽게 튜브를 몇 번 반전. 부드럽게 깨끗 한 75 µ m 나일론 메쉬 위에 소화 되지 않은 잎 조직 제거를 50ml 튜브에 효소/protoplasts 솔루션을 붓으십시오.</li>
	<li>실 온에서 1-2 분 50 mL 튜브에 100 x g에서 흐름을 통해 효소/protoplasts 솔루션 원심 부드럽게 상쾌한 원생 동물 펠 렛을 방해 하지 않고 10 mL 혈 청 학적인 피 펫을 사용 하 여 제거 합니다.</li>
	<li>Resuspend는 protoplasts 그리고 (x10) 현미경 hemacytometer에 원생 동물 수를 계산 하 여 4 ° C에서 냉장된 W5 솔루션에서 2 x 105 mL-1 의 농도를 희석. 30 분 동안 얼음에 protoplasts를 유지.</li>
	<li>실 온에서 1-2 분 x 100g에 현 탁 액을 원심 그리고 W5 솔루션 원생 동물 펠 렛을 방해 하지 않고 1 mL 피 펫을 사용 하 여 제거. 실 온에서 2 x 105 mL-1 의 농도에 MMG 솔루션 (표 1)에서 protoplasts를 resuspend.</li>
</ol>4. 원생 동물 전이
<ol><li>1.5 mL 낮은 접착 microcentrifuge 튜브 포경된 200 µ L 피 펫 팁을 사용 하 여 protoplasts (2 x 105 mL-1에서 2 × 104 protoplasts)의 100 µ L aliquots를 확인 합니다. 부정적인 통제 역할을 한 약 수 옆으로 넣어. 각 protoplasts의 나머지 약 수로 플라스 미드 (10-20 µ g)의 10 µ L를 추가 합니다.</li>
	<li>천천히 1.5 mL microcentrifuge 튜브의 내부 벽에 갓된 못 솔루션 (표 1)의 110 µ L을 추가 하 고 부드럽게 반전 솔루션은 균질 됩니다 때까지 튜브를 회전.</li>
	<li>15 분 동안 실 온에서 변환 혼합물을 품 어.</li>
	<li>중지 하려면 변환, 천천히 실내 온도에 1.5 mL 튜브에 W5 솔루션의 400 µ L을 추가 하 고 솔루션은 균질 됩니다 때까지 부드럽게 튜브를 반전 합니다. 실 온에서 1-2 분 100 g에 튜브 원심 하 고 상쾌한을 피 펫을 사용 하 여 삭제.</li>
	<li>튜브를 무선 솔루션 (표 1)의 1 mL을 추가 하 고 1-2 회 부드럽게 pipetting으로 resuspend. 코트 표면 하 고는 protoplasts 접시에 집착 하는 것을 방지 하 6 잘 조직 배양 플레이트의 각 음에 5% (vol/vol) 메 마른 송아지 혈 청 1 mL를 추가 합니다.</li>
	<li>몇 초 후, 송아지 혈 청을 피 펫을 사용 하 여 삭제 합니다. Resuspended protoplasts 문화 접시의 우물으로 전송 합니다. 커버 뚜껑 접시.</li>
</ol>5. 원생 동물 부 화 및 수확
<ol><li>어둠 속에서 1-2 일 동안 실 온에서 protoplasts를 품 어. 참고: 우리는 2 일 보육 일일 보육에 비해 일반적으로 더 강한 형광 단백질 신호 하 고 형광 단백질 신호 최대 3-4 일 동안 지속 될 수 있습니다 발견.</li>
	<li>원생 동물 솔루션 낮은 접착 microcentrifuge 1.5 mL 튜브에 전송. Protoplasts 수확을 실 온에서 1-2 분 x 100g에 튜브 원심 상쾌한 한 피 펫을 사용 하 여 제거 하 고 유리 슬라이드에 10 µ L protoplasts를 전송.</li>
	<li>Confocal 현미경 검사 법 또는 형광 형광 신호를 관찰 합니다. 변형 비 protoplasts를 사용 하 여 부정적인 제어 (신호가 관찰 한다).</li>
</ol>

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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>

저자 들은 아무 경쟁 금융 관심사 선언 합니다.

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.

<table><tbody><tr><td>MES</td><td>Sigma Aldrich</td><td>M8250-100G</td><td></td></tr><tr><td>Cellulase CELF</td><td>Worthington Biological Corporation</td><td>LS002611</td><td></td></tr><tr><td>Pectolyase Y-23</td><td>BioWorld</td><td>9033-35-6</td><td></td></tr><tr><td>CELLULASE &quot;ONOZUKA&quot; R-10</td><td>yakult</td><td>10g</td><td></td></tr><tr><td>MACEROZYME R-10</td><td>yakult</td><td>10g</td><td></td></tr><tr><td>Mannitol</td><td>ICN Biomedicals</td><td>152540</td><td></td></tr><tr><td>CaCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Fisher</td><td>C79-500g</td><td></td></tr><tr><td>BSA</td><td>NEB</td><td>R3535S</td><td></td></tr><tr><td>DTT</td><td>Sigma Aldrich</td><td>D5545-5G</td><td></td></tr><tr><td>NaCl</td><td>Sigma Aldrich</td><td>S7653-1kg</td><td></td></tr><tr><td>KCl</td><td>Fisher</td><td>P217-500g</td><td></td></tr><tr><td>MgCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Sigma Aldrich</td><td>M8266-100g</td><td></td></tr><tr><td>PEG4000</td><td>Fluka</td><td>81240</td><td></td></tr><tr><td>nylon mesh</td><td>carolina</td><td>652222N</td><td></td></tr><tr><td>Tissue Culture Plates</td><td>USA Scientific</td><td>CC7682-7506</td><td></td></tr><tr><td>Razor Blades</td><td>Fisher</td><td>12-640</td><td></td></tr><tr><td>hemacytometer</td><td>hausserscientific</td><td>1483</td><td></td></tr><tr><td>QIAprep Spin Miniprep Kit</td><td>Qiagen</td><td>27104</td><td></td></tr><tr><td>EZNA plasmid miniprep kit</td><td>Omega</td><td>D6942-01</td><td></td></tr><tr><td>GeneJET Plasmid Miniprep Kit</td><td>Thermo Scientific</td><td>K0502</td><td></td></tr><tr><td>Centrifuge 5810</td><td>eppendorf</td><td>5811000827</td><td></td></tr><tr><td>Centrifuge 5424</td><td>eppendorf</td><td>22620401</td><td></td></tr><tr><td>Jencons Powerpette Plus Pipet Controller</td><td>Jencons</td><td>14526-202</td><td></td></tr><tr><td>Zeiss 710 Confocal Microscope</td><td>Zeiss</td><td>N/A</td><td></td></tr><tr><td>Nonstick, RNase-free Microfuge Tubes, 1.5 mL</td><td>Ambion</td><td>AM12450</td><td></td></tr><tr><td>15 mL Centrifuge Tubes</td><td>Denville</td><td>C1018-P</td><td></td></tr><tr><td>50 mL Centrifuge Tubes</td><td>Denville</td><td>C1060-P</td><td></td></tr><tr><td>Newborn Calf Serum</td><td>Thermo Scientific</td><td>16010159</td><td></td></tr><tr><td>Soil</td><td>Ingram&#039;s Nursery</td><td></td><td></td></tr><tr><td>perlite</td><td>Vigoro</td><td>100521091</td><td></td></tr><tr><td>Torpedo Sand</td><td>JKS Ventures</td><td></td><td></td></tr><tr><td>LB Broth, Lennox (Powder)</td><td>Fisher</td><td>BP1427-500</td><td></td></tr></tbody></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. ...

a-simple-method-for-isolation-soybean-protoplasts-application-to

Research

JoVE Journal

Biology

24.6K 조회수.  California State University, Northridge. 우리는 라이브 셀에 복잡 한 규제 및 신호 메커니즘을 연구를 많은 양의 콩 protoplasts의 준비를 위한 간단 하 고 효율적인 프로토콜을 개발.

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

A Robotic Platform for High-throughput Protoplast Isolation and Transformation

Over the last decade there has been a resurgence in the use of plant protoplasts that range from model species to crop species, for analysis of signal transduction pathways, transcriptional regulatory networks, gene expression, genome-editing, and gene-silencing. Furthermore, significant progress has been made in the regeneration of plants from protoplasts, which has generated even more interest in the use of these systems for plant genomics. In this work, a protocol has been developed for automation of protoplast isolation and transformation from a &#39;Bright Yellow&#39; 2 (BY-2) tobacco suspension culture using a robotic platform. The transformation procedures were validated using an orange fluorescent protein (OFP) reporter gene (pporRFP) under the control of the Cauliflower mosaic virus 35S promoter (35S). OFP expression in protoplasts was confirmed by epifluorescence microscopy. Analyses also included protoplast production efficiency methods using propidium iodide. Finally, low-cost food-grade enzymes were used for the protoplast isolation procedure, circumventing the need for lab-grade enzymes that are cost-prohibitive in high-throughput automated protoplast isolation and analysis. Based on the protocol developed in this work, the complete procedure from protoplast isolation to transformation can be conducted in under 4 hr, without any input from the operator. While the protocol developed in this work was validated with the BY-2 cell culture, the procedures and methods should be translatable to any plant suspension culture/protoplast system, which should enable acceleration of crop genomics research.

A high-throughput, automated, tobacco protoplast production and transformation methodology is described. The robotic system enables massively parallel gene expression and discovery in the model BY-2 system that should be translatable to non-model crops.

높은 처리량 원형질체 분리 및 변환을위한 로봇 플랫폼

Over the last decade there has been a resurgence in the use of plant protoplasts that range from model species to crop species, for analysis of signal ...

연구

유전학

Transient Gene Expression in Tobacco using Gibson Assembly and the Gene Gun

In order to target a single protein to multiple subcellular organelles, plants typically duplicate the relevant genes, and express each gene separately using complex regulatory strategies including differential promoters and/or signal sequences. Metabolic engineers and synthetic biologists interested in targeting enzymes to a particular organelle are faced with a challenge: For a protein that is to be localized to more than one organelle, the engineer must clone the same gene multiple times. This work presents a solution to this strategy: harnessing alternative splicing of mRNA. This technology takes advantage of established chloroplast and peroxisome targeting sequences and combines them into a single mRNA that is alternatively spliced. Some splice variants are sent to the chloroplast, some to the peroxisome, and some to the cytosol. Here the system is designed for multiple-organelle targeting with alternative splicing. In this work, GFP was expected to be expressed in the chloroplast, cytosol, and peroxisome by a series of rationally designed 5&#8217; mRNA tags. These tags have the potential to reduce the amount of cloning required when heterologous genes need to be expressed in multiple subcellular organelles. The constructs were designed in previous work11, and were cloned using Gibson assembly, a ligation independent cloning method that does not require restriction enzymes. The resultant plasmids were introduced into Nicotiana benthamiana epidermal leaf cells with a modified Gene Gun protocol. Finally, transformed leaves were observed with confocal microscopy.

This work describes a novel method for selectively targeting subcellular organelles in plants, assayed using the BioRad Gene Gun.

깁슨 조립 및 유전자 총을 사용하여 담배 일시적인 유전자 발현

In order to target a single protein to multiple subcellular organelles, plants typically duplicate the relevant genes, and express each gene separately ...

환경과학

Isolation of Protoplasts from Tissues of 14-day-old Seedlings of Arabidopsis thaliana

Protoplasts are plant cells that have had their cell walls enzymatically removed. Isolation of protoplasts from different plant tissues was first reported more than 40 years ago 1 and has since been adapted to study a variety of cellular processes, such as subcellular localization of proteins, isolation of intact organelles and targeted gene-inactivation by double stranded RNA interference (RNAi) 2-5. Most of the protoplast isolation protocols use leaf tissues of mature Arabidopsis (e.g. 35-day-old plants) 2-4. We modified existing protocols by employing 14-day-old Arabidopsis seedlings. In this procedure, one gram of 14-day-old seedlings yielded 5 106-107 protoplasts that remain intact at least 96 hours. The yield of protoplasts from seedlings is comparable with preparations from leaves of mature Arabidopsis, but instead of 35-36 days, isolation of protoplasts is completed in 15 days. This allows decreasing the time and growth chamber space that are required for isolating protoplasts when mature plants are used, and expedites the downstream studies that require intact protoplasts.

Isolation of Protoplasts from Tissues of 14-day-old Seedlings of Arabidopsis thaliana

This video shows a procedure for isolating intact protoplasts from tissues of 14-day-old seedlings of Arabidopsis. Given that the isolated protoplasts remain intact for at least 96h and are isolated from seedlings instead of one-month-old mature plants, this procedure expedites assays requiring intact protoplasts.

14 일 된 모종의 조직에서 Protoplasts의 분리 Arabidopsis thaliana</em

Protoplasts are plant cells that have had their cell walls enzymatically removed. Isolation of protoplasts  from different plant tissues was first ...

생물학

Robotics and Dynamic Image Analysis for Studies of Gene Expression in Plant Tissues

Gene expression in plant tissues is typically studied by destructive extraction of compounds from plant tissues for in vitro analyses. The methods presented here utilize the green fluorescent protein (gfp) gene for continual monitoring of gene expression in the same pieces of tissues, over time. The gfp gene was placed under regulatory control of different promoters and introduced into lima bean cotyledonary tissues via particle bombardment. Cotyledons were then placed on a robotic image collection system, which consisted of a fluorescence dissecting microscope with a digital camera and a 2-dimensional robotics platform custom-designed to allow secure attachment of culture dishes. Images were collected from cotyledonary tissues every hour for 100 hours to generate expression profiles for each promoter. Each collected series of 100 images was first subjected to manual image alignment using ImageReady to make certain that GFP-expressing foci were consistently retained within selected fields of analysis. Specific regions of the series measuring 300 x 400 pixels, were then selected for further analysis to provide GFP Intensity measurements using ImageJ software. Batch images were separated into the red, green and blue channels and GFP-expressing areas were identified using the threshold feature of ImageJ. After subtracting the background fluorescence (subtraction of gray values of non-expressing pixels from every pixel) in the respective red and green channels, GFP intensity was calculated by multiplying the mean grayscale value per pixel by the total number of GFP-expressing pixels in each channel, and then adding those values for both the red and green channels. GFP Intensity values were collected for all 100 time points to yield expression profiles. Variations in GFP expression profiles resulted from differences in factors such as promoter strength, presence of a silencing suppressor, or nature of the promoter. In addition to quantification of GFP intensity, the image series were also used to generate time-lapse animations using ImageReady. Time-lapse animations revealed that the clear majority of cells displayed a relatively rapid increase in GFP expression, followed by a slow decline. Some cells occasionally displayed a sudden loss of fluorescence, which may be associated with rapid cell death. Apparent transport of GFP across the membrane and cell wall to adjacent cells was also observed. Time lapse animations provided additional information that could not otherwise be obtained using GFP Intensity profiles or single time point image collections.

We report a method for introduction, tracking and quantitative analysis of GFP expression in plant cells. This method utilizes a custom-designed robotics system for semi-continuous image collection from large numbers of samples, over time. We also demonstrate the use of ImageJ and ImageReady for analysis of image series.

로보틱스 및 식물 조직에서 유전자 발현의 연구 동적 이미지 분석

Gene expression in plant tissues is typically studied by destructive extraction of compounds from plant tissues for in vitro analyses. The methods ...

Investigating Tissue- and Organ-specific Phytochrome Responses using FACS-assisted Cell-type Specific Expression Profiling in Arabidopsis thaliana

Light mediates an array of developmental and adaptive processes throughout the life cycle of a plant. Plants utilize light-absorbing molecules called photoreceptors to sense and adapt to light. The red/far-red light-absorbing phytochrome photoreceptors have been studied extensively. Phytochromes exist as a family of proteins with distinct and overlapping functions in all higher plant systems in which they have been studied1. Phytochrome-mediated light responses, which range from seed germination through flowering and senescence, are often localized to specific plant tissues or organs2. Despite the discovery and elucidation of individual and redundant phytochrome functions through mutational analyses, conclusive reports on distinct sites of photoperception and the molecular mechanisms of localized pools of phytochromes that mediate spatial-specific phytochrome responses are limited. We designed experiments based on the hypotheses that specific sites of phytochrome photoperception regulate tissue- and organ-specific aspects of photomorphogenesis, and that localized phytochrome pools engage distinct subsets of downstream target genes in cell-to-cell signaling. We developed a biochemical approach to selectively reduce functional phytochromes in an organ- or tissue-specific manner within transgenic plants. Our studies are based on a bipartite enhancer-trap approach that results in transactivation of the expression of a gene under control of the Upstream Activation Sequence (UAS) element by the transcriptional activator GAL43. The biliverdin reductase (BVR) gene under the control of the UAS is silently maintained in the absence of GAL4 transactivation in the UAS-BVR parent4. Genetic crosses between a UAS-BVR transgenic line and a GAL4-GFP enhancer trap line result in specific expression of the BVR gene in cells marked by GFP expression4. BVR accumulation in Arabidopsis plants results in phytochrome chromophore deficiency in planta5-7. Thus, transgenic plants that we have produced exhibit GAL4-dependent activation of the BVR gene, resulting in the biochemical inactivation of phytochrome, as well as GAL4-dependent GFP expression. Photobiological and molecular genetic analyses of BVR transgenic lines are yielding insight into tissue- and organ-specific phytochrome-mediated responses that have been associated with corresponding sites of photoperception4, 7, 8. Fluorescence Activated Cell Sorting (FACS) of GFP-positive, enhancer-trap-induced BVR-expressing plant protoplasts coupled with cell-type-specific gene expression profiling through microarray analysis is being used to identify putative downstream target genes involved in mediating spatial-specific phytochrome responses. This research is expanding our understanding of sites of light perception, the mechanisms through which various tissues or organs cooperate in light-regulated plant growth and development, and advancing the molecular dissection of complex phytochrome-mediated cell-to-cell signaling cascades.

Investigating Tissue- and Organ-specific Phytochrome Responses using FACS-assisted Cell-type Specific Expression Profiling in Arabidopsis thaliana

The molecular basis of spatial-specific phytochrome responses is being investigated using transgenic plants that exhibit tissue- and organ-specific phytochrome deficiencies. The isolation of specific cells exhibiting induced phytochrome chromophore depletion by Fluorescence-Activated Cell Sorting followed by microarray analyses is being utilized to identify genes involved in spatial-specific phytochrome responses.

에서 외과를 이용한 셀 타입 특정 표현식 프로파일을 사용하여 조직 및 장기 특정 Phytochrome 응답 조사 Arabidopsis thaliana</em

Light mediates an array of developmental and adaptive processes throughout the life cycle of a plant. Plants utilize light-absorbing molecules called ...

Identification of Plasmodesmal Localization Sequences in Proteins In Planta

Plasmodesmata (Pd) are cell-to-cell connections that function as gateways through which small and large molecules are transported between plant cells. Whereas Pd transport of small molecules, such as ions and water, is presumed to occur passively, cell-to-cell transport of biological macromolecules, such proteins, most likely occurs via an active mechanism that involves specific targeting signals on the transported molecule. The scarcity of identified plasmodesmata (Pd) localization signals (PLSs) has severely restricted the understanding of protein-sorting pathways involved in plant cell-to-cell macromolecular transport and communication. From a wealth of plant endogenous and viral proteins known to traffic through Pd, only three PLSs have been reported to date, all of them from endogenous plant proteins. Thus, it is important to develop a reliable and systematic experimental strategy to identify a functional PLS sequence, that is both necessary and sufficient for Pd targeting, directly in the living plant cells. Here, we describe one such strategy using as a paradigm the cell-to-cell movement protein (MP) of the Tobacco mosaic virus (TMV). These experiments, that identified and characterized the first plant viral PLS, can be adapted for discovery of PLS sequences in most Pd-targeted proteins.

Identification of Plasmodesmal Localization Sequences in Proteins In Planta

Plant intercellular connections, the plasmodesmata (Pd), play central roles in plant physiology and plant-virus interactions. Critical to Pd transport are sorting signals that direct proteins to Pd. However, our knowledge about these sequences is still in its infancy. We describe a strategy to identify Pd localization signals in Pd-targeted proteins.

Plasmodesmal 지역화 시퀀스 Planta에 단백질의 식별

Plasmodesmata (Pd) are cell-to-cell connections that function as gateways through which small and large molecules are transported between plant cells. ...

면역학 및 감염병학

Efficient and Rapid Isolation of Early-stage Embryos from Arabidopsis thaliana Seeds

In flowering plants, the embryo develops within a nourishing tissue - the endosperm - surrounded by the maternal seed integuments (or seed coat). As a consequence, the isolation of plant embryos at early stages (1 cell to globular stage) is technically challenging due to their relative inaccessibility. Efficient manual dissection at early stages is strongly impaired by the small size of young Arabidopsis seeds and the adhesiveness of the embryo to the surrounding tissues. Here, we describe a method that allows the efficient isolation of young Arabidopsis embryos, yielding up to 40 embryos in 1 hr to 4 hr, depending on the downstream application. Embryos are released into isolation buffer by slightly crushing 250-750 seeds with a plastic pestle in an Eppendorf tube. A glass microcapillary attached to either a standard laboratory pipette (via a rubber tube) or a hydraulically controlled microinjector is used to collect embryos from droplets placed on a multi-well slide on an inverted light microscope. The technical skills required are simple and easily transferable, and the basic setup does not require costly equipment. Collected embryos are suitable for a variety of downstream applications such as RT-PCR, RNA sequencing, DNA methylation analyses, fluorescence in situ hybridization (FISH), immunostaining, and reporter gene assays.

Efficient and Rapid Isolation of Early-stage Embryos from Arabidopsis thaliana Seeds

We report an efficient and simple method to isolate embryos at early stages of development from Arabidopsis thaliana seeds. Up to 40 embryos can be isolated in 1 hr to 4 hr, depending on the downstream application. The procedure is suitable for transcriptome, DNA methylation, reporter gene expression, immunostaining and fluorescence in situ hybridization analyses.

에서 초기 단계의 배아의 효율적이고 신속한 분리<em&gt; 애기 장대</em&gt; 씨앗

In flowering plants, the embryo develops within a nourishing tissue - the endosperm - surrounded by the maternal seed integuments (or seed coat). As a ...

형질전환 대두 털 뿌리의 고효율 생산

Soybean Hairy Root Transformation for the Analysis of Gene Function

Soybean (Glycine max) is a valuable crop in agriculture that has thousands of industrial uses. Soybean roots are the primary site of interaction with soil-borne microbes that form symbiosis to fix nitrogen and pathogens, which makes research involving soybean root genetics of prime importance to improve its agricultural production. The genetic transformation of soybean hairy roots (HRs) is mediated by the Agrobacterium rhizogenes strain NCPPB2659 (K599) and is an efficient tool for studying gene function in soybean roots, taking only 2 months from start to finish. Here, we provide a detailed protocol that outlines the method for overexpressing and silencing a gene of interest in soybean HRs. This methodology includes soybean seed sterilization, infection of cotyledons with K599, and the selection and harvesting of genetically transformed HRs for RNA isolation and, if warranted, metabolite analyses. The throughput of the approach is sufficient to simultaneously study several genes or networks and could determine the optimal engineering strategies prior to committing to long-term stable transformation approaches.

저자 스포트라이트: 유전자 기능 분석을 위한 대두 털 뿌리 형질전환  

Here, we present a protocol for the high-efficiency production of transgenic soybean hairy roots.

유전자 기능 분석을 위한 콩 털이 뿌리 형질전환

Soybean (Glycine max) is a valuable crop in agriculture that has thousands of industrial uses. Soybean roots are the primary site of interaction with ...

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

요약

더 많은 비디오 탐색

이 비디오의 챕터

높은 처리량 원형질체 분리 및 변환을위한 로봇 플랫폼

깁슨 조립 및 유전자 총을 사용하여 담배 일시적인 유전자 발현

14 일 된 모종의 조직에서 Protoplasts의 분리 Arabidopsis thaliana

로보틱스 및 식물 조직에서 유전자 발현의 연구 동적 이미지 분석

에서 외과를 이용한 셀 타입 특정 표현식 프로파일을 사용하여 조직 및 장기 특정 Phytochrome 응답 조사 Arabidopsis thaliana

Plasmodesmal 지역화 시퀀스 Planta에 단백질의 식별

에서 초기 단계의 배아의 효율적이고 신속한 분리

유전자 기능 분석을 위한 콩 털이 뿌리 형질전환

유전자 기능 분석을 위한 콩 털이 뿌리 형질전환

유전자 기능 분석을 위한 콩 털이 뿌리 형질전환