도움이 의견을 거스 - 절대 안전한 프로토콜 Paja Sijacic과 코트니 Hollender를 공유하기위한 거스와 그림 1, Detlef 위겔의 사진을 : 우리는 pTSO2 구축을위한 Chunxin 왕 감사합니다. Z. L S 실험실에서 연구는 미국 국립 과학 재단 (IOB0616096 및 MCB0744752)에 의해 지원됩니다. ZL는 부분적으로 메릴랜드 농업 실험 역 대학에 의해 지원됩니다.

I. 꽃 - 물놀이 Arabidopsis thaliana의 변화 <ol><li> 이전 변환에 약 1 개월, 냄비에 약 10-15 식물 4-8 냄비 (5 인치 사각형 냄비)에 Arabidopsis 씨를 뿌리다. 정상 다산과 식물, 플라스미드 구조마다 식물의 4 냄비는 충분합니다. 하나는 감소 다산과 돌연변이 Arabidopsis를 변환하는 경우, 그에 따라 냄비의 수를 증가시킵니다. </li><li> 식물은 표준 약관 (22 16 시간 조명과 어두운 8 시간 ° C)에서 재배하고 있습니다. 식물은 짧은 하루 더 낮은 온도 하에서 재배하는 경우, 그들은 변화를 위해 준비하는 꽃 이상으로 오래 걸립니다. 좋은 식물 관리는 성공적인 변화를위한 필수적인 건강한 식물을 보장합니다. </li><li> 식물은 나올와 꽃으로 시작하는 경우. 그들은 지금 변화를위한 준비가되어 있습니다. 변환 효율을 향상시키기 위해, 3-4일 변환하기 전에, 곧 그들은 더 많은 보조 촬영 형성을 장려하기 위해 나올 필요로 메인 꽃이 핌 골인을 잘라. 식물 변환 전날 물. 이것은 토양이 꽃의 수영하는 동안 너무 많이 침투 미디어를 흡수하지 않을 보장합니다. </li><li> Agrobacterium tumefaciens 변형 GV3101은 pTSO2를 들고와 (거스, 50 μg / ML Kanamycin : pTSO2의 경우) : 이틀 전에 변환하기 위해, 적절한 항생제를 포함한 40ml LB의 예방 : 거스는 건설. pTSO2 : : 거스는 Kanamycin 3 저항을 수여 NPTII 유전자를 가지고 pBIN20 벡터에 복제됩니다. 28-30 ° C.에 떨고 배양기에서 하룻밤 성장 </li><li> 그 다음날, 50 μg / ML Kanamycin이 포함된 LB의 400ml에 밤새 문화의 전송 8ml. 28-30 ° C.에 떨고 배양기에서 하룻밤 성장 </li><li> 변화의 날, Agrobacterium 문화의 OD 600 약 0.8 (0.5 to1 사이 OD 600 허용된다)에 도달하면, 5000rpm 8 분에서 Agrobacterium 야간 문화를 스핀 다운. </li><li> 침투 미디어 1 L에있는 Agrobacterium 펠렛을 (리터) (10mM MgCl 2, 5 %의 자당, 0.44mM 6 benzyladenine (BA), 0.3 % silwet L - 77, 1X Gamborg의 비타민 솔루션과 autoclaved 물). Resuspend 매체 autoclaved해야하지만 신선한 만들 필요하지 않습니다. 접시 (예 : 8로 &quot;x15&quot;X2 &quot;Pyrex 유리 접시)에 1 L Agrobacterium 함유 침투 매체를 붓고. </li><li> 냄비 반전 (식물에서 떨어지지 않을 것이란)와 침투 솔루션에 식물의 모든 공중 부분을 찍어, 한 번에 한 오분 냄비 기다. 공장의 모든 항공 부분이 침투 매체에 열중하는 동안, 토양은 토양에 곰팡이 성장의 기회를 줄이기 위해 침투 미디어 중 하나를 흠뻑 젖어셔서 마십시오. </li><li> 마른 후, 트레이에 종이 타월의 여러 레이어에서 자신의 측면에서 모든 담근 판돈을 놓으십시오. 종이 타올은 침투 미디어의 접근 금액을 만끽해보세요. 높은 습도를 보장하기 위해 플라스틱 포장으로 트레이를 다룹니다. 식물 성장 챔버에 트레이를 놓습니다. </li><li> 다음날, 똑바로 플라스틱 랩, 종이​​ 수건, 장소의 냄비를 제거합니다. 4-5 더 많은 일 동안이 식물을 물을 마십시오. 이후, 그들은 씨앗을 설정할 때까지 정상적으로 식물을 유지하고 대량의 종자를 수집합니다. </li><li> 4g의 멋진 한천, 압력솥, 그리고 추가 1M NaOH와 5.8-500 ML의 물, 산도에 용해, MS 매체가 50 μg / ML Kanamycin (중량 2.2g Murashige가 포함된 비타민없이 기초 소금을 Skoog와 함께 접시 (150x15mm 페트리 접시)을 부어 ) 50 μg / ML 최종 농도 Kanamycin를 추가합니다. 번호판이 필요 ° C까지 4 보관됩니다. </li><li> 항생제 - 내성 유전자 변형 식물을 선택하기 위해, 하나는 최소한 20,000 씨앗을 화면으로 필요합니다. 0.1 그램은 약 5,000 씨앗입니다. 무게와 씨앗의 양을 예상됩니다. </li><li> 30 초 동안 70 %의 에탄올과 15 ML 팰컨 튜브를 세척하여 씨를 소독. 한번 무균 물로 씨앗을 씻으십시오. 가게에서 구입 5 % 표백제 (Clorox)의 1시 10분 희석이 들어있는 용액에 30 초 동안 씨앗을 만끽해보세요. 멸균 물 3-6 시간을 씨를 린스. 무균 후드, 각 MS (Kanamycin) 플레이트에 피펫 5000에 대한 씨앗은 오염을 피하기 위해 Micropore 3M ​​테이프로 번호판을 봉인, 접시에 균일하게 씨앗을 확산. </li><li> 4 번호판을 품어 ° C 3-4 일 동안 어둠 속에서, 다음 챔버 (같은 식물 성장 챔버 수)에 접시를 전송합니다. 약 14 일 후, 유전자 변형 모종 녹색 유지하지만 이외의 유전자 변형 것들은 창백 돌려 죽어 가고 있습니다. 둔화가 매체에서 뿌리를 철수하고 토양에서 그들을 배치하여 토양에 유전자 변형 묘목을 전송합니다. </li></ol> II. pTSO2을 검토 : : 거스 표현 패턴 <ol><li> 수확 pTSO2 : 거스 형질 전환 모종 및 유리 섬광 약병이나 microfuge 튜브의 감기 90 % 아세톤에 배치그것은 얼음에 보관됩니다. 한 샘플의 큰 숫자를 분석하는 경우 폴리스티렌 microtiter 플레이트 (안 폴리 프로필렌)도 사용할 수 있습니다. </li><li> 모든 샘플이 채취 후 20 분 상온에서 튜브를 놓으십시오. 이 시간 동안 X - Gluc (0.2 % 트리톤 X - 100, 50mM NaHPO 4 버퍼 pH7.2, 2mM 칼륨 Ferrocyanide, 2mM의 칼륨 Ferricyanide)와 얼음에 장소없이 신선한 얼룩 버퍼를합니다. </li><li> 샘플에서 아세톤을 제거하고 얼룩 버퍼를 추가합니다. </li><li> X - Gluc 얼룩 솔루션 (0.2 % 트리톤 X - 100, 50mM NaHPO 4 버퍼 pH7.2, 2mM의 칼륨 Ferrocyanide, 2mM의 칼륨 Ferricyanide, 2mM X - gluc)를 구성합니다. 샘플에서 얼룩 버퍼를 제거하고 샘플을 X - Gluc을 포함하고있는 얼룩 버퍼를 추가합니다. </li><li> 15-20분위한 진공 아래 샘플을 침투. 천천히 진공을 해제하고 모든 샘플이 얼룩 솔루션의 표면 아래 싱크되었는지 확인합니다. 필요한 경우 진공이 릴리스되면 모든 샘플 싱크까지 침투를 반복합니다. </li><li> 37 품어 샘플 ° C X - Gluc와 얼룩 버퍼에 하룻밤. </li><li> 인큐베이터에서 샘플을 제거하고 얼룩 버퍼를 제거합니다. 삼십분마다 변경 상온에서 (20 %, 35 % 및 50 % 에탄올) 연속 에탄올 시리즈의 샘플을 씻으십시오. </li><li> 실온에서 30 분 FAA 정착액 (50 % 에탄올, 5 % 포름 알데히드 10 % 초산, 나머지 물)에 조직을 수정. FAA를 제거하고 70 % 에탄올을 추가합니다. 이 시점에서 조직은 시각 수 있으며 디지털 카메라가 장착된 해부 현미경 사진. TSO2가 (그림 1) 표현이 어디 진한 파란색 색상을 나타냅니다. 또한, 조직은 ° C 나중에 볼 수 있도록 4 저장할 수 있습니다. <img src="/files/ftp_upload/1952/1952fig1.jpg" alt="그림 1" /> 그림 1 pTSO2 :. : 유전자 변형 모종의 거스 표현 (A), 측면 뿌리 (B), 그리고 루트 팁 (C). TSO2 발기인은 젊은 분열 조직에서 매우 활성화되어 있습니다. (C)와 같이 산발적 표현식 패턴은 특정 세포주기의 단계에있는 유전자 표현의 전형이다. </li></ol> 대표 결과 제대로하면, 변환 효율은 약 0.1-0.2 %되어야합니다. 다른 단어에서, 하나는 모든 5,000 종자를 선별하여 50-10 형질 모종을해야합니다. 평균 약 100 유전자 변형 라인은 야생 형 식물의 4 냄비를 변환하여 얻을 수 있습니다. 에 대한 pTSO2를 포함하는 유전자 변형 모종 : 거스 구축 3, 거스 활동을 반영 진한 파란색 색상 어린 나뭇잎, 쏴, 아펙스, 루트 팁, 그리고 측면 루트 primordia (그림 1)을 포함하여 적극적으로 나누어 세포에서 발견된다. 비 균일한 산발적 패턴은 세포주기 단계 - 구체적인 표현의 특징입니다.

<ol>
	<li>Clough, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Floral+dip:+agrobacterium-mediated+germ+line+transformation.">Floral dip: agrobacterium-mediated germ line transformation.</a> Methods Mol Biol. 286, 91-91 (2005).</li><li>Clough, S. J., Bent, A. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Floral+dip:+a+simplified+method+for+Agrobacterium-mediated+transformation+of+Arabidopsis+thaliana.">Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana.</a> Plant J. 16 (6), 735-735 (1998).</li><li>Wang, C., Liu, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Arabidopsis+ribonucleotide+reductases+are+critical+for+cell+cycle+progression,+DNA+damage+repair,+and+plant+development.">Arabidopsis ribonucleotide reductases are critical for cell cycle progression, DNA damage repair, and plant development.</a> Plant Cell. 18 (2), 350-350 (2006).</li></ol>

관심 없음 충돌 선언하지 않습니다.

변환 효율은 아래 설명하는 여러 가지 요인에 의해 결정됩니다 : <ol><li> 식물의 건강과 자신의 나이가 기본 중요합니다. 다수의 미숙 꽃 봉오리를 생산 약 한 달쯤 식물 이상적입니다. 변환하기 전에 보조 촬영 형성 3-4일 권장하는 기본 골인을 트리밍하는 것은 변환 효율을 증가합니다. 이전 식물 transformants을 일으키다하지만 낮은 비율로합니다. </li><li> 식물의 불임은 중요하다. 하나는 감소 다...

<table><tbody><tr><td>6-benzyladenine</td><td>Sigma-Aldrich</td><td>852430</td><td></td></tr><tr><td>Silwet-77</td><td>Crompton Corp.</td><td></td><td>Toxic, wear gloves</td></tr><tr><td>Murashige and Skoog Basal medium (MS)</td><td>Sigma-Aldrich</td><td>M5519</td><td></td></tr><tr><td>Gamborg&amp;rsquo;s vitamin solution 1000X</td><td>Sigma-Aldrich</td><td>G1019</td><td></td></tr><tr><td>X-Gluc (5-bromo-4-chloro-3-indolyl &amp;#946;-D-glucuronide cyclohexamine salt</td><td>Biosynth International, Inc</td><td>B-7300</td><td>Toxic, light sensitive keep in the dark</td></tr><tr><td>Micropore 3M Tape</td><td>Fisher Scientific</td><td>19027761</td><td></td></tr></tbody></table>

floral-dip transformation of arabidopsis thaliana to examine ptso2::&beta;-glucuronidase reporter gene expression

유기체로 외국 유전자를 도입하는 능력은 현대 생물학과 생명 공학에 대한 기초입니다. 모델 꽃 공장에서<em&gt; Arabidopsis thaliana</em&gt; 꽃 - 딥 변환 방법<sup&gt; 1-2</sup&gt; 때문에 간단, 효율성 및 저렴한 비용의 이전의 모든 방법을 대체하고 있습니다. 젊은 꽃의 특히, 골인<em&gt; Arabidopsis</em&gt; 식물의 솔루션에 담근 아르<em&gt; Agrobacterium tumefaciens</em&gt; 구체적인 플라스미드 구조를 들고. 마른 후, 식물이 정상적인 성장과 항복 씨앗, 외국 유전자로 변환하고 항생제를 포함하는 매체에 대해 선택할 수있는 작은 비율로 반환됩니다. 크게 촉진이 꽃 - 물놀이 방법<em&gt; Arabidopsis</em&gt; 연구와 식물 유전자 기능에 대한 우리의 이해에 크게 공헌했습니다. 본 연구에서는, 우리는 기자의 유전자를 변형하는 꽃 - 수영 방법을 사용<em&gt; β - glucuronidase (거스)</em&gt;의 통제하에<em&gt; TSO2</em&gt; 발기인.<em&gt; TSO2</em&gt; Ribonucleotide 환원 효소 (RNR) 작은 subunit에 대한 코딩<sup&gt; 3</sup&gt; 세포주기의 S - 단계에서 dNDP 생합성에 필수적인 세포주기 규제 유전자입니다. 심사<em&gt; 거스</em유전자 변형&gt;의 표현<em&gt; Arabidopsis</em&gt; 모종가를 보여줍니다<em&gt; TSO2</em&gt;를 적극적으로 조직을 나누어 표현입니다. 보고된 실험 방법 및 자료를 쉽게 고등학교 및 대학 수준의 연구뿐만 아니라 교육뿐만 아니라 적용할 수 있습니다.

Watch this Scientific Journal Video about Floral-dip Transformation of Arabidopsis thaliana to Examine pTSO2::Î²-glucuronidase Reporter Gene Expression at JoVE.com

Floral-dip Transformation of Arabidopsis thaliana to Examine pTSO2::&#946;-glucuronidase Reporter Gene Expression

이 문서의 꽃 - 수영 방법을 보여줍니다<em&gt; Agrobacterium tumefaciens</em의&gt; - 중재 변환<em&gt; Arabidopsis thaliana</em&gt;. 세포주기 조절 발기인 - 기자를 소개함으로써,<em&gt; pTSO2 : β - glucuronidase (거스)</em&gt;로<em&gt; Arabidopsis</em&gt; 우리는 하나가 유전자 변형 모종의 거스 기자 표현식을 감지하는 방법을 보여줍니다.

의 꽃 - 물놀이 변환 Arabidopsis thaliana 검사 pTSO2 : β - glucuronidase 리포터 유전자 발현

The ability to introduce foreign genes into an organism is the foundation for modern biology and biotechnology. In the model flowering plant Arabidopsis ...

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Floral-dip Transformation of Arabidopsis thaliana to Examine pTSO2::&beta;-glucuronidase Reporter Gene Expression

36.1K 조회수.  University of Maryland College Park. 이 문서의 꽃 - 수영 방법을 보여줍니다 Agrobacterium tumefaciens - 중재 변환 Arabidopsis thaliana. 세포주기 조절 발기인 - 기자를 소개함으로써, pTSO2 : β - glucuronidase (거스)로 Arabidopsis 우리는 하나가 유전자 변형 모종의 거스 기자 표현식을 감지하는 방법을 보여줍니다.

비디오: Floral-dip Transformation of Arabidopsis thaliana to Examine pTSO2::β-glucuronidase Reporter Gene Expression

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 ...

연구

환경과학

Generating Transgenic Plants with Single-copy Insertions Using BIBAC-GW Binary Vector

When generating transgenic plants, generally the objective is to have stable expression of a transgene. This requires a single, intact integration of the transgene, as multi-copy integrations are often subjected to gene silencing. The Gateway-compatible binary vector based on bacterial artificial chromosomes (pBIBAC-GW), like other pBIBAC derivatives, allows the insertion of single-copy transgenes with high efficiency. As an improvement to the original pBIBAC, a Gateway cassette has been cloned into pBIBAC-GW, so that the sequences of interest can now be easily incorporated into the vector transfer DNA (T-DNA) by Gateway cloning. Commonly, the transformation with pBIBAC-GW results in an efficiency of 0.2&#8211;0.5%, whereby half of the transgenics carry an intact single-copy integration of the T-DNA. The pBIBAC-GW vectors are available with resistance to Glufosinate-ammonium or DsRed fluorescence in seed coats for selection in plants, and with resistance to kanamycin as a selection in bacteria. Here, a series of protocols is presented that guide the reader through the process of generating transgenic plants using pBIBAC-GW: starting from recombining the sequences of interest into the pBIBAC-GW vector of choice, to plant transformation with Agrobacterium, selection of the transgenics, and testing the plants for intactness and copy number of the inserts using DNA blotting. Attention is given to designing a DNA blotting strategy to recognize single- and multi-copy integrations at single and multiple loci.

Using a pBIBAC-GW binary vector makes generating transgenic plants with intact single-copy insertions, an easy process. Here, a series of protocols is presented that guide the reader through the process of generating transgenic Arabidopsis plants, and testing the plants for intactness and copy number of the inserts.

BIBAC-GW 이진 벡터를 사용 하 여 단일 복사본 삽입을 가진 유전자 변형 식물 생성

When generating transgenic plants, generally the objective is to have stable expression of a transgene. This requires a single, intact integration of the ...

유전학

Inducible, Cell Type-Specific Expression in Arabidopsis thaliana Through LhGR-Mediated Trans-Activation

Inducible, tissue-specific expression is an important and powerful tool to study the spatio-temporal dynamics of genetic perturbation. Combining the flexible and efficient GreenGate cloning system with the proven and benchmarked LhGR system (here termed GR-LhG4) for the inducible expression, we have generated a set of transgenic Arabidopsis lines that can drive the expression of an effector cassette in a range of specific cell types in the three main plant meristems. To this end, we chose the previously developed GR-LhG4 system based on a chimeric transcription factor and a cognate pOp-type promoter ensuring tight control over a wide range of expression levels. In addition, to visualize the expression domain where the synthetic transcription factor is active, an ER-localized mTurquoise2 fluorescent reporter under control of the pOp4 or pOp6 promoter is encoded in driver lines. Here, we describe the steps necessary to generate a driver or effector line and demonstrate how cell type specific expression can be induced and followed in the shoot apical meristem, the root apical meristem and the cambium of Arabidopsis. By using several or all driver lines, the context specific effect of expressing one or multiple factors (effectors) under control of the synthetic pOp promoter can be assessed rapidly, for example in F1 plants of a cross between one effector and multiple driver lines. This approach is exemplified by the ectopic expression of VND7, a NAC transcription factor capable of inducing ectopic secondary cell wall deposition in a cell autonomous manner.

Inducible, Cell Type-Specific Expression in Arabidopsis thaliana Through LhGR-Mediated Trans-Activation

Here, we describe the generation and application of a set of transgenic Arabidopsis thaliana lines enabling inducible, tissue-specific expression in the three main meristems, the shoot apical meristem, the root apical meristem, and the cambium.

유도할 수 있는, 애기 thaliana 통해 LhGR 중재 트랜스에 셀 형식 관련 식-활성화

Inducible, tissue-specific expression is an important and powerful tool to study the spatio-temporal dynamics of genetic perturbation. Combining the ...

Co-expression of Multiple Chimeric Fluorescent Fusion Proteins in an Efficient Way in Plants

Information about the spatiotemporal subcellular localization(s) of a protein is critical to understand its physiological functions in cells. Fluorescent proteins and generation of fluorescent fusion proteins have been wildly used as an effective tool to directly visualize the protein localization and dynamics in cells. It is especially useful to compare them with well-known organelle markers after co-expression with the protein of interest. Nevertheless, classical approaches for protein co-expression in plants usually involve multiple independent expression plasmids, and therefore have drawbacks that include low co-expression efficiency, expression-level variation, and high time expenditure in genetic crossing and screening. In this study, we describe a robust and novel method for co-expression of multiple chimeric fluorescent proteins in plants. It overcomes the limitations of the conventional methods by using a single expression vector that is composed of multiple semi-independent expressing cassettes. Each protein expression cassette contains its own functional protein expression elements, and therefore it can be flexibly adjusted to meet diverse expression demand. Also, it is easy and convenient to perform the assembly and manipulation of DNA fragments in the expression plasmid by using an optimized one-step reaction without additional digestion and ligation steps. Furthermore, it is fully compatible with current fluorescent protein derived bio-imaging technologies and applications, such as FRET and BiFC. As a validation of the method, we employed this new system to co-express fluorescently fused vacuolar sorting receptor and secretory carrier membrane proteins. The results show that their perspective subcellular localizations are the same as in previous studies by both transient expression and genetic transformation in plants.

We have developed a novel method for co-expressing multiple chimeric fluorescent fusion proteins in plants to overcome the difficulties of conventional methods. It takes advantage of using a single expression plasmid that contains multiple functionally independent protein expressing cassettes to achieve protein co-expression.

식물에는 효율적인 방법으로 여러 공상 형광 성 융해 단백질의 공동 식

Information about the spatiotemporal subcellular localization(s) of a protein is critical to understand its physiological functions in cells. Fluorescent ...

화학

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 ...

생물학

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

Cell Specific Analysis of Arabidopsis Leaves Using Fluorescence Activated Cell Sorting

After initiation of the leaf primordium, biomass accumulation is controlled mainly by cell proliferation and expansion in the leaves1. However, the Arabidopsis leaf is a complex organ made up of many different cell types and several structures. At the same time, the growing leaf contains cells at different stages of development, with the cells furthest from the petiole being the first to stop expanding and undergo senescence1. Different cells within the leaf are therefore dividing, elongating or differentiating; active, stressed or dead; and/or responding to stimuli in sub-sets of their cellular type at any one time. This makes genomic study of the leaf challenging: for example when analyzing expression data from whole leaves, signals from genetic networks operating in distinct cellular response zones or cell types will be confounded, resulting in an inaccurate profile being generated.
To address this, several methods have been described which enable studies of cell specific gene expression. These include laser-capture microdissection (LCM)2 or GFP expressing plants used for protoplast generation and subsequent fluorescence activated cell sorting (FACS)3,4, the recently described INTACT system for nuclear precipitation5 and immunoprecipitation of polysomes6.
FACS has been successfully used for a number of studies, including showing that the cell identity and distance from the root tip had a significant effect on the expression profiles of a large number of genes3,7. FACS of GFP lines have also been used to demonstrate cell-specific transcriptional regulation during root nitrogen responses and lateral root development8, salt stress9 auxin distribution in the root10 and to create a gene expression map of the Arabidopsis shoot apical meristem11. Although FACS has previously been used to sort Arabidopsis leaf derived protoplasts based on autofluorescence12,13, so far the use of FACS on Arabidopsis lines expressing GFP in the leaves has been very limited4. In the following protocol we describe a method for obtaining Arabidopsis leaf protoplasts that are compatible with FACS while minimizing the impact of the protoplast generation regime. We demonstrate the method using the KC464 Arabidopsis line, which express GFP in the adaxial epidermis14, the KC274 line, which express GFP in the vascular tissue14 and the TP382 Arabidopsis line, which express a double GFP construct linked to a nuclear localization signal in the guard cells (data not shown; Figure 2). We are currently using this method to study both cell-type specific expression during development and stress, as well as heterogeneous cell populations at various stages of senescence.

Cell Specific Analysis of Arabidopsis Leaves Using Fluorescence Activated Cell Sorting

A method for producing Arabidopsis leaf protoplasts that are compatible with fluorescence activated cell sorting (FACS), allowing for studies of specific cell populations. This method is compatible with any Arabidopsis line that expresses GFP in a subset of cells.

의 세포 구체적인 분석<em&gt; Arabidopsis</em&gt; 형광 활성화 셀 정렬을 사용하여 잎

After initiation of the leaf primordium, biomass accumulation is controlled mainly by cell proliferation and expansion in the leaves1. However, the ...

Floral-Dip Transformation of Flax (Linum usitatissimum) to Generate Transgenic Progenies with a High Transformation Rate

Agrobacterium-mediated plant transformation via floral-dip is a widely used technique in the field of plant transformation and has been reported to be successful for many plant species. However, flax (Linum usitatissimum) transformation by floral-dip has not been reported. The goal of this protocol is to establish that Agrobacterium and the floral-dip method can be used to generate transgenic flax. We show that this technique is simple, inexpensive, efficient, and more importantly, gives a higher transformation rate than the current available methods of flax transformation.
In summary, inflorescences of flax were dipped in a solution of Agrobacterium carrying a binary vector plasmid (T-DNA fragment plus the Linum Insertion Sequence, LIS-1) for 1 - 2 min. The plants were laid flat on their side for 24 hr. Then, plants were maintained under normal growth conditions until the next treatment. The process of dipping was repeated 2 - 3 times, with approximately 10 - 14 day intervals between dipping. The T1 seeds were collected and germinated on soil. After approximately two weeks, treated progenies were tested by direct PCR; 2 - 3 leaves were used per plant plus the appropriate T-DNA primers. Positive transformants were selected and grown to maturity. The transformation rate was unexpectedly high, with 50 - 60% of the seeds from treated plants being positive transformants. This is a higher transformation rate than those reported for Arabidopsis thaliana and other plant species, using floral-dip transformation. It is also the highest, which has been reported so far, for flax transformation using other methods for transformation.

Floral-Dip Transformation of Flax Linum usitatissimum to Generate Transgenic Progenies with a High Transformation Rate

Here, we present a protocol to transform flax using Agrobacterium-mediated plant transformation via floral-dip. This protocol is simple to perform and inexpensive, yet yields a higher transformation rate than the current available methods for flax transformation.

아마의 꽃 - 딥 변환 (<em&gt; LINUM usitatissimum</em&gt;) 높은 변환 속도와 형질 전환 자손을 생성합니다

Agrobacterium-mediated plant transformation via floral-dip is a widely used technique in the field of plant transformation and has been reported to be ...

Rapid Analysis of Circadian Phenotypes in Arabidopsis Protoplasts Transfected with a Luminescent Clock Reporter

The plant circadian clock allows the anticipation of daily changes to the environment. This anticipation aids the responses to temporally predictable biotic and abiotic stress. Conversely, disruption of circadian timekeeping severely compromises plant health and reduces agricultural crop yields. It is therefore imperative that we understand the intricate regulation of circadian rhythms in plants, including the factors that affect motion of the transcriptional clockwork itself.
Testing circadian defects in the model plant Arabidopsis thaliana (Arabidopsis) traditionally involves crossing specific mutant lines to a line rhythmically expressing firefly luciferase from a circadian clock gene promoter. This approach is laborious, time-consuming, and could be fruitless if a mutant has no circadian phenotype. The methodology presented here allows a rapid initial assessment of circadian phenotypes. Protoplasts derived from mutant and wild-type Arabidopsis are isolated, transfected with a rhythmically expressed luminescent reporter, and imaged under constant light conditions for 5 days. Luminescent traces will directly reveal whether the free-running period of mutant plants is different from wild-type plants. The advantage of the method is that any Arabidopsis line can efficiently be screened, without the need for generating a stably transgenic luminescent clock marker line in that mutant background.

The circadian clock regulates about a third of the Arabidopsis transcriptome, but the percentage of genes that feed back into timekeeping remains unknown. Here we visualize a method to rapidly assess circadian phenotypes in any mutant line of Arabidopsis using luminescent imaging of a circadian reporter transiently expressed in protoplasts.

발광 시계 기자와 애기 장대 원형질체 트랜의 일주기 표현형의 신속한 분석

The plant circadian clock allows the anticipation of daily changes to the environment. This anticipation aids the responses to temporally predictable ...

A Versatile Method for Mounting Arabidopsis Leaves for Intravital Time-lapse Imaging

The plant immune response associated with a genome-wide transcriptional reprogramming is initiated at the site of infection. Thus, the immune response is regulated spatially and temporally. The use of a fluorescent gene under the control of an immunity-related promoter in combination with an automated fluorescence microscopy is a simple way to understand spatiotemporal regulation of plant immunity. In contrast to the root tissues that have been used for a number of various intravital fluorescent imaging experiments, there exist few fluorescent live-imaging examples for the leaf tissues that encounter an array of airborne microbial infections. Therefore, we developed a simple method to mount leaves of Arabidopsis thaliana plants for live-cell imaging over an extended period of time. We used transgenic Arabidopsis plants expressing the yellow fluorescent protein (YFP) genefused to the nuclear localization signal (NLS) under the control of the promoter of a defense-related marker gene, Pathogenesis-Related 1 (PR1). We infiltrated a transgenic leaf with Pseudomonas syringae pv. tomato DC3000 (avrRpt2) strain (Pst_a2) and performed in vivo time-lapse imaging of the YFP signal for a total of 40 h using an automated fluorescence stereomicroscope. This method can be utilized not only for studies on plant immune responses but also for analyses of various developmental events and environmental responses occurring in leaf tissues.

A Versatile Method for Mounting Arabidopsis Leaves for Intravital Time-lapse Imaging

We report a simple and versatile method for performing fluorescent live-imaging of Arabidopsis thaliana leaves over an extended period of time. We use a transgenic Arabidopsis plant expressing a fluorescent reporter gene under the control of an immunity-related promoter as an example for gaining spatiotemporal understanding of plant immune responses.

Intravital 시간 경과 영상에 대 한 애기 를 장착 하는 다양 한 방법을 나뭇잎합니다

The plant immune response associated with a genome-wide transcriptional reprogramming is initiated at the site of infection. Thus, the immune response is ...

의 꽃 - 물놀이 변환 Arabidopsis thaliana 검사 pTSO2 : β - glucuronidase 리포터 유전자 발현

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