Bu eser bitki genom araştırma programı Ulusal Bilim Vakfı (NSF-PGRP-IOS-1339388) tarafından desteklenmiştir.

1. bitkilerin büyüme
<ol><li>5-10 soya tohumları (Williams 82) özel toprak karışımı üzerinde 25 ° c (16 h 1500 µmol m-2 s-1ışık) uzun günlük koşullarda sera içinde 13 cm tencerede soya için ekmek (1:1:1 oran toprak, perlit ve torpido kum).</li>
</ol>2. plazmid DNA hazırlanması
<ol><li>Steril pipet ucu veya kürdan kullanarak, bir koloni veya faiz gen içeren plazmid taşıyan E. coli donmuş gliserol hisse senedi almak ve 20 mL Luria-Bertani (LB) sıvı ortamda bir 50 mL şişe olarak uygun antibiyotik aşılamak.</li>
	<li>Gecede bir shaker olarak 37 ° C'de şişeye kuluçkaya. Bakteri süspansiyon 12.000 x g 5 min için oda sıcaklığında, centrifuging ve süpernatant atarak toplamak. Hulâsa ve plazmid hazırlık seti üreticinin prosedürü takip plazmid arındırmak.</li>
</ol>3. protoplast yalıtım
<ol><li>10 - gün eski soya fidan (şekil 1) yeni genişletilmiş unifoliate yaprakları kesti. Not: Yaprakları uygun bir gelişimsel aşamada başarının anahtarı soya protoplast hazırlık için seçimdir. Sadece sadece genişletilmiş yaprakları aşamalarında erken gelişimsel kullanın. Olgun yaprakları, hücre duvarları daha zor sindirmek için hale gelir.</li>
	<li>Taze bir tıraş bıçağı ile midrib unifoliate yaprak ve kalıntıları içine 0,5-1 mm şeritler kes çıkarın. Not: iki unifoliate yaprakları 10 mL enzim çözümde sindirmek için 10'dan fazla dönüşümleri yeterli önceki verecektir.</li>
	<li>Forseps, transfer bir çift kullanarak yaprak 10 mL taze hazırlanmış enzim çözeltisi (Tablo 1) bir 15 mL tüp içine hemen ve yavaşça şeritler. Vakum infiltrat yaprak 15 dakika oda sıcaklığında şeritler.</li>
	<li>Yaprak şeritler ile nazik ajitasyon (40 d/d) düşük ışık altında oda sıcaklığında 4-6 h için enzim çözümde kuluçkaya. Önceki yayımlanan olarak enzim çözüm sarı-yeşil döner emin olun. Enzim/önceki çözüm (X10) mikroskop altında denetleyin. Not: Yayımlanan önceki küresel şeklinde, sindirilmemiş hücreler düzensiz veya oval şekli varken.</li>
	<li>Yavaşça dökerek enzim/protoplast çözüm bir 50 mL tüp 10 mL transfer ve hazım durdurmak için oda sıcaklığında 10 mL W5 çözeltisi (Tablo 1) ekleyin. Yavaşça tüp birkaç kez tersine çevirin. Yavaşça sindirilmemiş yaprak dokuları kaldırmak için 50 mL tüp üzerine yerleştirilen bir temiz 75 µm naylon mesh üzerinde enzim/önceki solüsyonu dökün.</li>
	<li>100 x g oda sıcaklığında 1-2 dk 50 mL tüp, akışı sayesinde enzim/önceki çözüm santrifüj kapasitesi. Yavaşça protoplast Pelet bozmadan bir 10 mL serolojik pipet kullanarak süpernatant çıkarın.</li>
	<li>Önceki resuspend ve (x10) mikroskop altında bir hemacytometer numaralı protoplast sayarak 2 x 105 mL-1 4 ° C'de soğutulmuş W5 çözümde bir konsantrasyon sulandırmak. Önceki 30 dk için buz üzerinde tutun.</li>
	<li>100 x g oda sıcaklığında 1-2 min için de süspansiyon santrifüj kapasitesi ve 1 mL pipet protoplast Pelet bozmadan kullanarak W5 çözüm yavaşça çıkarın. 2 x 105 mL-1 oda sıcaklığında bir konsantrasyon, MMG çözüm (Tablo 1) önceki resuspend.</li>
</ol>4. protoplast dönüştürme
<ol><li>100 µL aliquots önceki (2 x 104 önceki vasıl 2 x 105 mL-1), 1.5 mL düşük yapışma microcentrifuge tüplere kesilmemiş 200 µL pipet ipuçlarını kullanarak olun. Negatif denetimi hizmet için bir aliquot bir kenara koyun. Plazmid (10-20 µg) 10 µL her önceki dinlenme aliquot içine ekleyin.</li>
	<li>Yavaş taze hazırlanmış PEG çözüm (Tablo 1) 110 µL 1.5 mL microcentrifuge tüp iç duvarında ekleyin ve sonra yavaşça tersine çevirin ve çözüm homojen hale gelinceye kadar tüp döndürün.</li>
	<li>Dönüştürme karışımı 15 dakika oda sıcaklığında kuluçkaya.</li>
	<li>Dönüşümün durdurmak için yavaş yavaş 1,5 mL tüp, oda sıcaklığında 400 µL W5 çözüm ekleyin ve çözüm homojen hale gelinceye kadar tüpü yavaşça tersine çevirin. Tüp 100 gr oda sıcaklığında 1-2 min için santrifüj kapasitesi ve bir pipet kullanarak süpernatant atın.</li>
	<li>WI çözüm (Tablo 1) 1 mL tüp ekleyin ve hafifçe 1 - 2 defa pipetting tarafından resuspend. 1 mL % 5 (vol/vol) steril buzağı serum her şey yüzey kat ve önceki plakasına yapışmasını önlemek için 6-iyi doku kültürü plaka ekleyin.</li>
	<li>Birkaç saniye sonra bir pipet kullanarak buzağı serum atmak. Resuspended önceki kültür plaka bir kuyunun içine aktarın. Bir kapak ile plaka kapak.</li>
</ol>5. protoplast kuluçka ve hasat
<ol><li>1-2 gün içinde belgili tanımlık karanlık oda sıcaklığında önceki kuluçkaya. Not: Bulduğumuz iki günde kuluçka genel olarak bir günlük kuluçka için karşılaştırıldığında güçlü floresan protein sinyali verir ve floresan protein sinyal 3-4 güne kadar sürebilir.</li>
	<li>Protoplast çözüm 1.5 mL düşük yapışma microcentrifuge tüp aktarın. Önceki hasat için oda sıcaklığında 1-2 min için 100 x g de tüp santrifüj kapasitesi. Bir pipet kullanarak süpernatant kaldırmak ve 10 µL önceki bir cam slayt üzerine aktarın.</li>
	<li>Floresan veya confocal mikroskobu altında floresan sinyal gözlemlemek. Sigara dönüştürülmüş önceki (sinyal yok dikkat edilmelidir) negatif kontrol kullanın.</li>
</ol>

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Yazarlar onlar rakip hiçbir mali çıkarları var bildirin.

Bu iletişim kuralı soya önceki ve geçici ifade çalışmaları uygulama yalıtım için kapsamlı biçimde sınanmış ve çok iyi bizim laboratuarında çalışıyor. Yordamlar basit ve kolay ve sıradan ekipman ve minimum maliyet gerektirir. Bizim protokol Tekdüzen, yüksek kaliteli önceki daha önce bildirilen yöntemler34,35,36,37,38için karşılaş...

Önceki hücre duvarlarının kaldırılması içeren bitki hücreleri vardır. Onlar özelliklerin çoğunu ve bitki hücreleri faaliyetlerinin sürdürdükçe, önceki gözlemlemek ve çeşitli hücresel olaylar değerlendirmek için bir iyi model sistemi vardır ve somatik melezleştirme1 çalışma ve rejenerasyon2bitki değerli araçlardır. Hücre duvarları aksi halde DNA hücre içine geçişini engellemek istiyorsunuz bu yana önceki bitki dönüşümü3,4,5için aynı zamanda yaygın olarak kullanılan. Önceki bazı fizyolojik tepkilerin ve dolayısıyla hücre altı protein Yerelleştirme6,7,8çalışmaya temel araştırma temel değeri sunan sağlam bitkilerin hücresel süreçler sahip, protein-protein etkileşimleri9,10ve organizatörü etkinliği11,12,13 ' te hücreleri yaşıyor.
Bitki önceki yalıtım ilk 196014 ' te bildirildi ve protokolleri yalıtım ve önceki dönüşümü için geliştirilen ve en iyi duruma getirilmiş. Standart bir prosedür protoplast tecrit yaprakların kesme ve Enzimatik sindirim yayımlanan önceki ayrılması tarafından sindirilmiş sigara doku enkazı takip hücre duvarlarının içerir. Dönüşüm stratejileri elektroporasyon15,16, mikroenjeksiyon17,18ve polietilen glikol tabanlı (PEG)4,5,19 yöntemleri içerir. Türlerin geniş bildirilmiştir protoplast yalıtımı, narenciye20, Brassica21, Solanaceae22 ve diğer süs bitki aileler23,24gibi başarılı. Çeşitli doku tipleri çeşitli türler ise, Arabidopsis mesophyll işgal (TEAMP) geçici ifade Arabidopsis thaliana modeli bitki yapraklarından izole bir sistem iyi kurulmuş25 oldu ve çeşitli uygulamalar için yaygın olarak kabul edilen.
Soya (Glycine max (L.) Merr'e.) bir en önemli protein ve yağ26kırpar. Arabidopsis ve pirinç aksine, transgenik soya bitkiler elde etmek oldukça zor ve düşük verimlilik olduğu bilinmektedir. Agrobacterium tumefaciens-aracılı infiltrasyon halk kullanılan tütün27 ' deki epidermal hücrelerin ve fidan Arabidopsis28,29, geçici gen ifade araştırmaları için ise Agrobacterium rhizogenes kıllı soya30kökleri dönüştürme için kullanılmıştır. Virüs kaynaklı gene yaklaşımlar susturmak için hedef genlerin31,32 ve geçici ifade33 downregülasyon sistemik bir şekilde kullanılmıştır. Önceki bu yaklaşımlar için değerli ve çok yönlü bir alternatif sağlar. Önceki soya'nın yerüstü malzemelerden elde edilebilir ve hızlı ve senkronize transgene ifade sağlar. Ancak, soya önceki341983 yılından bu yana ilk başarılı yalıtım sınırlı rapor edilmiştir önceki soya35,36,37, ve uygulamaya 38, öncelikle soya önceki nispeten düşük verimleri nedeniyle.
Burada, soya önceki yalıtım için basit ve etkili iletişim kuralı ve uygulama geçici gen ifade Etütler açıklayın. Soya fidan genç unifoliate yaprakları kullanarak, biz büyük miktarda hayati önceki birkaç saat içinde elde edebildik. Buna ek olarak, basit ve düşük maliyetli DNA soya önceki yüksek verimlilik ile teslim etmek PEG-kalsiyum-aracılı dönüşüm yöntemi optimize.

<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

Soya (Glycine max (L.) Merr'e.) bir önemli bitki türü ve genetik ve biyokimyasal yolları araştırmalar baklagil modeli haline geldi. Bu nedenle, soya içinde bir verimli geçici gen ifade sistemi kurmak önemlidir. Burada, soya önceki hazırlanması için basit bir protokol ve geçici işlevsel analizleri için uygulama raporu. Bulduk soya fidan genç unifoliate yaprakları kaliteli önceki büyük miktarlarda sonuçlandı. PEG-kalsiyum-aracılı dönüşüm yöntemi optimize ederek, soya unifoliate önceki kullanarak yüksek dönüşüm verimliliği elde etti. Bu sistem etkin ve çok yönlü modeli karmaşık düzenleyici ve sinyal mekanizmaları incelenmesi canlı soya hücrelerdeki için sağlar ve Bakliyat çeşitli hücresel, gelişimsel ve fizyolojik süreçleri anlamak zorunda yardım için daha iyi.

10 - gün eski soya fasulyesi farklı organları işgal hazırlık (şekil 1) için test edildi ve verimleri (Şekil 2) mikroskop altında gözlendi. Hücre duvarları hypocotyl ve epicotyl zor sindirmek ve bazı hücreler birbirine (2B şekil, 2 C) bağlı kaldı. Cotyledon (şekil 2B) ve kök (şekil 2A), hücre duvarları...

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

Biz canlı hücrelerdeki karmaşık düzenleyici ve sinyal mekanizmaları incelemek soya önceki büyük miktarlarda hazırlanması için basit ve etkili iletişim kuralı geliştirilmiştir.

Soya önceki ve geçici gen ifade analiz için uygulama yalıtım için basit bir yöntem

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

Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

24.6K Görüntüleme.  California State University, Northridge. Biz canlı hücrelerdeki karmaşık düzenleyici ve sinyal mekanizmaları incelemek soya önceki büyük miktarlarda hazırlanması için basit ve etkili iletişim kuralı geliştirilmiştir.

Video: 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.

Yüksek verim Protoplast İzolasyonu ve Dönüşüm için Robotik Platformu

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 ...

Genetik

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.

Gibson Meclisi ve Gen Tabancası kullanarak Tütün geçici Gen İfadesi

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

Çevre

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 gün eski Fideler Dokular protoplast izolasyonu Arabidopsis thaliana</em

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

Biyoloji

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.

Bitkisel Dokular Gen İfade Araştırmalar Robotik ve Dinamik Görüntü Analizi

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.

Hücre tipi Özgül gösterilmeden FACS yardımlı kullanarak Doku ve Organ spesifik Fitokrom Yanıtları incelenmesi 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.

Proteinlerin içinde Planta serilerinde Plasmodesmal yerelleştirme tanımlaması

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

İmmünoloji ve Enfeksiyon

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.

Erken evre Embriyolar verimli ve hızlı İzolasyon<em&gt; Arabidopsis thaliana</em&gt; Tohumlar

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

Transgenik Soya Fasulyesi Tüylü Köklerinin Yüksek Verimli Üretimi

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.

Yazar Spotlight: Gen Fonksiyonunun Analizi için Soya Fasulyesi Tüylü Kök Dönüşümü  

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

Gen fonksiyonunun analizi için soya fasulyesi tüylü kök transformasyonu

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

Soya önceki ve geçici gen ifade analiz için uygulama yalıtım için basit bir yöntem

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