<ol>
	<li>ASTM D1413-07. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Standard+test+method+for+testing+wood+preservatives+by+laboratory+soil-block+cultures.+.">Standard test method for testing wood preservatives by laboratory soil-block cultures. .</a> Annual Book of ASTM Standards. , 185-192 (2007).</li><li>Bravery, 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=.">.</a> A miniaturized wood block for the rapid evaluation of wood preservative fungicides. , (1978).</li><li>Low, G. A., Young, M. E., Martin, P., Palfreyman, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assessing+the+relationship+between+the+dry+rot+fungus+Serpula+lacrymans+and+selected+forms+of+masonry.">Assessing the relationship between the dry rot fungus Serpula lacrymans and selected forms of masonry.</a> Int. Biodeterior. Biodegrad. 46, 141-150 (2000).</li><li>Nicolas, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Volume+I+(One/1)+-+Degradation+and+Protection+of+Wood+(Syracuse+Wood+Science+Series+#5).">Volume I (One/1) - Degradation and Protection of Wood (Syracuse Wood Science Series #5).</a> Wood Deterioration and Its Prevention by Preservative Treatments. , (1973).</li><li>Schilling, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+calcium-based+materials+and+iron+impurities+on+wood+degradation+by+the+brown+rot+fungus+Serpula+lacrymans.">Effects of calcium-based materials and iron impurities on wood degradation by the brown rot fungus Serpula lacrymans.</a> Holzforschung. 64, 93-99 (2010).</li></ol>

관심 없음 충돌 선언하지 않습니다.

우리 한천 블록 설정 (그림 1)를 사용 Serpula lacrymans은 (그림 3 제어 갈색 - 부패 소나무 블록에 60 % 이상의 체중 감소로 이어지는, 석고 표면과 나무 블록 (그림 2)에 직접 접촉하는 성장 ). 이것은 쉽게&gt; 50 %의 부패의 ASTM 표준 목표를 만족하고,에서 부패에 변화의 평균 계수 (C V)는 주 16 0.055했다. 이 데이터는 실링 7 게시됩니다. 다시 다른 곰팡이는 토양 블록보다 한천 블록에...

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Petri dishes</td>
<td>&nbsp;</td>
<td>Nunc</td>
<td>4014</td>
<td>25 x 150 mm</td>
</tr>
<tr>
<td>Agar, Type A</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>A4550</td>
<td>&nbsp;</td>
<tr>
<td>Ammonium nitrate, NH4NO3</td>
<td>&nbsp;</td>
<td>Millinckrodt</td>
<td>3436-12</td>
<td>&nbsp;</td>
<tr>
<td>Potassium phosphate, KH2PO4</td>
<td>&nbsp;</td>
<td>J.T. Baker</td>
<td>3246-01</td>
<td>&nbsp;</td>
<tr>
<td>Magnesium sulfate 7-hydrate, MgSO4&bull;7H2O</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>230391</td>
<td>&nbsp;</td>
<tr>
<td>D-(+)-Glucose</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>G8270</td>
<td>Dextrose</td>
</tr>
<tr>
<td>Boric acid, H3BO4</td>
<td>&nbsp;</td>
<td>Mallinckrodt</td>
<td>2549-04</td>
<td>&nbsp;</td>
<tr>
<td>Zinc sulfate 7-hydrate, ZnSO4&bull;7H2O</td>
<td>&nbsp;</td>
<td>Mallinckrodt</td>
<td>8880-12</td>
<td>&nbsp;</td>
<tr>
<td>Manganous chloride 4-hydrate, MnCl2&bull;4H2O</td>
<td>&nbsp;</td>
<td>J.T. Baker</td>
<td>2540-04</td>
<td>&nbsp;</td>
<tr>
<td>Copper(II) sulfate 5-hydrate, CuSO4&bull;5H2O</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>209198</td>
<td>&nbsp;</td>
<tr>
<td>Ammonium heptamolybdate 4-hydrate, (NH4)6Mo7O24&bull;4H2O</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>431346</td>
<td>&nbsp;</td>
<tr>
<td>Calcium chloride dihydrate, CaCl2&bull;2H2O</td>
<td>&nbsp;</td>
<td>Mallinckrodt</td>
<td>4160-12</td>
<td>&nbsp;</td>
<tr>
<td>Sodium chloride, NaCl</td>
<td>&nbsp;</td>
<td>Mallinckrodt</td>
<td>7581-12</td>
<td>&nbsp;</td>
<tr>
<td>Ferrous sulfate 7-hydrate, FeSO4&bull;7H2O</td>
<td>&nbsp;</td>
<td>Mallinckrodt</td>
<td>5056-12</td>
<td>&nbsp;</td>
<tr>
<td>Pipet-aid</td>
<td>&nbsp;</td>
<td>Drummond</td>
<td>4-000-110</td>
<td>Cordless 
EtOH the surface</td>
</tr>
<tr>
<td>10 ml sterile polystyrene pipette</td>
<td>&nbsp;</td>
<td>BD Biosciences</td>
<td>357551</td>
<td>&nbsp;</td>
<tr>
<td>Gutter Guard</td>
<td>&nbsp;</td>
<td>Thermwell Products Co.</td>
<td>VX620</td>
<td>Pre-scrubbed with soap 
Hardware store</td>
</tr>
<tr>
<td>Calcium sulfate hemihydrate, CaSO4&bull;0.5H2O</td>
<td>&nbsp;</td>
<td>Acros Organics</td>
<td>385355000</td>
<td>&nbsp;</td>
<tr>
<td>#4 cork borer</td>
<td>&nbsp;</td>
<td>Boekel</td>
<td>1601</td>
<td>&nbsp;</td>
<tr>
<td>Parafilm "M"</td>
<td>&nbsp;</td>
<td>Pechiney</td>
<td>PM-996</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

agar-block microcosms for controlled plant tissue decomposition by aerobic fungi

리그노셀룰로오스성 식물 조직의 곰팡이 biodegradation를 공부에 대한 두 가지 주요 방법은 목재 방부제 테스트 (; 한천 블록 토양 블록)에 대한 개발되었습니다. 이것은 토양 블록 microcosms 높은 붕괴 속도, 적은 수분 문제, 연구 중 낮은 다양성, 그리고 방부제의 독성 높은 임계값을 얻을 것을 잘 허용됩니다. 토양 블록 테스트 따라서 더 활용 기법이며, 테스트 및 재료에 대한 미국 사회 (ASTM) (방법 D 1413-07)에 의해 표준화되었습니다. 토양 블록 디자인은 로컬 변수 토양 소스를 사용하지만, 단점을 가지고 있으며, 썩어 조직에 영양분을 외부 (외인성)의 통제를 제한 인치 이러한 단점은 다른, 점점 인기를 연구 목적으로이 방법을 적용의 문제로 등장했습니다. 이러한 현대의 목적은 네트워크 hyphal 함께 산화 메카니즘 및 추적 translocated 요소를 평가 공동 대사 toxics의 bioremediation 테스트 bioenergy 연구를위한 굴욕 lignocellulosics를 포함. 토양 - 블록은 이러한 응용 프로그램에 충분한 제어를 빌려하지 않습니다. 정제 한천 블록 접근 방식이 필요합니다. 여기, 우리는 나무 저하 곰팡이 Serpula lacrymans은 낮은 칼슘 한천과 함께 깊은 배양 접시를 이용하여 한천 블록 microcosms의 나무이 저하될 수있는 갈색 썩음을 사용합니다. 우리는 유틸리티 및 예상 다양성을 설명하는 시간 시리즈 부패에 대한 외인성 석고의 역할을 테스트합니다. 싱글 보드 격랑 (세로 컷)에서 블록은 냉방 무게, autoclaved, 플라스틱 메쉬 위에 무균 소개하고 있습니다. 외인성 석고는 인터페이스에서 추가로 곰팡이 예방 접종, 각 블록 얼​​굴을하고 있습니다. 수확은 최종 파괴 추수 때까지 무균 수 있습니다. 이러한 microcosms은 한천이나 배양 접시의 벽 블록 접촉을 피하기 위해 설계되었습니다. 결로는 접시 쏟아 도중 부화하는 동안 최소화합니다. 마지막으로, inoculum / 석고 / 목재 간격은 있지만 접촉을 허용하지 않고 최소화됩니다. 한천 블록 디자인이 덜 기술적인 관점은 또한 오류 및 연구 사이에 변화의 핵심 소스의 가장 흔한 원인입니다. 비디오 간행물이 경우에 따라서 유용합니다, 우리는 낮은 다양성, 높은 품질의 결과를 보여줍니다.

Watch this Scientific Journal Video about Agar-Block Microcosms for Controlled Plant Tissue Decomposition by Aerobic Fungi at JoVE.com

Agar-Block Microcosms for Controlled Plant Tissue Decomposition by Aerobic Fungi

이 동영상은 호기성 곰팡이로 리그노셀룰로오스성 식물 조직의 저하를 연구하기위한 통제된 환경의 접근 방식을 보여줍니다. 영양 소스 및 습기를 제어하는​​ 능력은 한천 블록 microcosms의 주요 장점이지만, 그 접근 방식은 종종 혼합 성공을 산출. 우리는 재현성, 낮은 다양성의 결과를 얻을 수에 중요한 함정을 해결합니다.

에어로빅 곰팡이에 의해 제어 플랜트 조직 분해에 대한 한천 - 블럭 Microcosms

The two principal methods for studying fungal biodegradation of lignocellulosic plant tissues were developed for wood preservative testing (soil-block; ...

agar-block-microcosms-for-controlled-plant-tissue-decomposition

Research

JoVE Journal

Biology

14.9K 조회수.  University of Minnesota. 이 동영상은 호기성 곰팡이로 리그노셀룰로오스성 식물 조직의 저하를 연구하기위한 통제된 환경의 접근 방식을 보여줍니다. 영양 소스 및 습기를 제어하는​​ 능력은 한천 블록 microcosms의 주요 장점이지만, 그 접근 방식은 종종 혼합 성공을 산출. 우리는 재현성, 낮은 다양성의 결과를 얻을 수에 중요한 함정을 해결합니다.

비디오: Agar-Block Microcosms for Controlled Plant Tissue Decomposition by Aerobic Fungi

Measuring Plant Cell Wall Extension (Creep) Induced by Acidic pH and by Alpha-Expansin

Growing plant cell walls characteristically exhibit a property known as 'acid growth', by which we mean they are more extensible at low pH (&lt; 5) 1. The plant hormone auxin rapidly stimulates cell elongation in young stems and similar tissues at least in part by an acid-growth mechanism 2, 3. Auxin activates a H+ pump in the plasma membrane, causing acidification of the cell wall solution. Wall acidification activates expansins, which are endogenous cell wall-loosening proteins 4, causing the cell wall to yield to the wall tensions created by cell turgor pressure. As a result, the cell begins to enlarge rapidly. This 'acid growth' phenomenon is readily measured in isolated (nonliving) cell wall specimens. The ability of cell walls to undergo acid-induced extension is not simply the result of the structural arrangement of the cell wall polysaccharides (e.g. pectins), but depends on the activity of expansins 5. Expansins do not have any known enzymatic activity and the only way to assay for expansin activity is to measure their induction of cell wall extension. This video report details the sources and preparation techniques for obtaining suitable wall materials for expansin assays and goes on to show acid-induced extension and expansin-induced extension of wall samples prepared from growing cucumber hypocotyls.
 To obtain suitable cell wall samples, cucumber seedlings are grown in the dark, the hypocotyls are cut and frozen at -80 &deg;C. Frozen hypocotyls are abraded, flattened, and then clamped at constant tension in a special cuvette for extensometer measurements. To measure acid-induced extension, the walls are initially buffered at neutral pH, resulting in low activity of expansins that are components of the native cell walls. Upon buffer exchange to acidic pH, expansins are activated and the cell walls extend rapidly. We also demonstrate expansin activity in a reconstitution assay. For this part, we use a brief heat treatment to denature the native expansins in the cell wall samples. These inactivated cell walls do not extend even in acidic buffer, but addition of expansins to the cell walls rapidly restores their ability to extend.

Measuring Plant Cell Wall Extension Creep Induced by Acidic pH and by Alpha-Expansin

We demonstrate the use of a constant-force extensometer to measure long-term extension (creep) of plant cell wall specimens induced by acidic buffers and expansin protein.

측정 식물 세포 벽 확장 (크리프)는 산성 산도에 의해 및 알파 Expansin에 의해 유도된

Growing plant cell walls characteristically exhibit a property known as 'acid growth', by which we mean they are more extensible at low pH (&lt; 5) 1. The ...

연구

생물학

Linking Predation Risk, Herbivore Physiological Stress and Microbial Decomposition of Plant Litter

The quantity and quality of detritus entering the soil determines the rate of decomposition by microbial communities as well as recycle rates of nitrogen (N) and carbon (C) sequestration1,2. Plant litter comprises the majority of detritus3, and so it is assumed that decomposition is only marginally influenced by biomass inputs from animals such as herbivores and carnivores4,5. However, carnivores may influence microbial decomposition of plant litter via a chain of interactions in which predation risk alters the physiology of their herbivore prey that in turn alters soil microbial functioning when the herbivore carcasses are decomposed6. A physiological stress response by herbivores to the risk of predation can change the C:N elemental composition of herbivore biomass7,8,9 because stress from predation risk increases herbivore basal energy demands that in nutrient-limited systems forces herbivores to shift their consumption from N-rich resources to support growth and reproduction to C-rich carbohydrate resources to support heightened metabolism6. Herbivores have limited ability to store excess nutrients, so stressed herbivores excrete N as they increase carbohydrate-C consumption7. Ultimately, prey stressed by predation risk increase their body C:N ratio7,10, making them poorer quality resources for the soil microbial pool likely due to lower availability of labile N for microbial enzyme production6. Thus, decomposition of carcasses of stressed herbivores has a priming effect on the functioning of microbial communities that decreases subsequent ability to of microbes to decompose plant litter6,10,11.
We present the methodology to evaluate linkages between predation risk and litter decomposition by soil microbes. We describe how to: induce stress in herbivores from predation risk; measure those stress responses, and measure the consequences on microbial decomposition. We use insights from a model grassland ecosystem comprising the hunting spider predator (Pisuarina mira), a dominant grasshopper herbivore (Melanoplus femurrubrum),and a variety of grass and forb plants9.

We present methods to evaluate how predation risk can alter the chemical quality of herbivore prey by inducing dietary changes to meet demands of heightened stress, and how the decomposition of carcasses from these stressed herbivores slows subsequent plant litter decomposition by soil microbes.

Predation 위험, Herbivore 생리적 스트레스와 식물 돼지 미생물 분해를 연결

The quantity and quality of detritus entering the soil  determines the rate of decomposition by microbial communities as well as  recycle rates of ...

Isolation of Viable Multicellular Glands from Tissue of the Carnivorous Plant, Nepenthes

Many plants possess specialized structures that are involved in the production and secretion of specific low molecular weight compounds and proteins. These structures are almost always localized on plant surfaces. Among them are nectaries or glandular trichomes. The secreted compounds are often employed in interactions with the biotic environment, for example as attractants for pollinators or deterrents against herbivores.
Glands that are unique in several aspects can be found in carnivorous plants. In so-called pitcher plants of the genus Nepenthes, bifunctional glands inside the pitfall-trap on the one hand secrete the digestive fluid, including all enzymes necessary for prey digestion, and on the other hand take-up the released nutrients. Thus, these glands represent an ideal, specialized tissue predestinated to study the underlying molecular, biochemical, and physiological mechanisms of protein secretion and nutrient uptake in plants. Moreover, generally the biosynthesis of secondary compounds produced by many plants equipped with glandular structures could be investigated directly in glands.
In order to work on such specialized structures, they need to be isolated efficiently, fast, metabolically active, and without contamination with other tissues. Therefore, a mechanical micropreparation technique was developed and applied for studies on Nepenthes digestion fluid. Here, a protocol is presented that was used to successfully prepare single bifunctional glands from Nepenthes traps, based on a mechanized microsampling platform. The glands could be isolated and directly used further for gene expression analysis by PCR techniques after preparation of RNA.

Isolation of Viable Multicellular Glands from Tissue of the Carnivorous Plant, Nepenthes

Plants glands are specialized structures responsible for the biosynthesis and secretion of many compounds involved in interactions with the biotic environment. To enable studies on their molecular and biochemical features, a mechanical micropreparation technique was established in order to isolate single metabolically active glands, here from the carnivorous plant Nepenthes.

Many plants possess specialized structures that are involved in the production and secretion of specific low molecular weight compounds and proteins. ...

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) maintain the turnover of all mature blood cell lineages. The coordination of the complex signals leading to specific HSC fates relies upon the interaction between HSCs and the intricate bone marrow microenvironment, which is still poorly understood[1-2].
We describe how by combining a newly developed specimen holder for stable animal positioning with multi-step confocal and two-photon in vivo imaging techniques, it is possible to obtain high-resolution 3D stacks containing HSPCs and their surrounding niches and to monitor them over time through multi-point time-lapse imaging. High definition imaging allows detecting ex vivo labeled hematopoietic stem and progenitor cells (HSPCs) residing within the bone marrow. Moreover, multi-point time-lapse 3D imaging, obtained with faster acquisition settings, provides accurate information about HSPC movement and the reciprocal interactions between HSPCs and stroma cells.
Tracking of HSPCs in relation to GFP positive osteoblastic cells is shown as an exemplary application of this method. This technique can be utilized to track any appropriately labeled hematopoietic or stromal cell of interest within the mouse calvarium bone marrow space.

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

The nature of the interactions between hematopoietic stem and progenitor cells (HSPCs) and bone marrow niches is poorly understood. Custom hardware modifications and a multi-step acquisition protocol allow the use of two-photon and confocal microscopy to image ex vivo labeled HSPCs homed within bone marrow areas, tracking interactions and movement.

성인 마우스 두개골 골수에서 조혈 줄기 및 전구 세포의 생체 내 4 차원 추적에서

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) ...

High-throughput CRISPR Vector Construction and Characterization of DNA Modifications by Generation of Tomato Hairy Roots

Targeted DNA mutations generated by vectors with clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 technology have proven useful for functional genomics studies. While most cloning strategies are simple to perform, they generally use multiple steps and can require several days to generate the ultimate constructs. The method presented here is based on DNA assembly and can produce fully functional CRISPR vectors in a single cloning reaction. Vector construction can also be pooled, further increasing the efficiency and utility of the process. A modification of the method is used to create CRISPR vectors with multiple gene targets. CRISPR vectors are then transformed into tomato hairy roots to generate transgenic materials with targeted DNA modifications. Hairy roots are a useful system for testing vector functionality as they are technically simple to generate and amenable to large-scale production. The methods presented here will have wide application as they can be used to generate a variety of CRISPR vectors and be used in a wide range of plant species.

Using DNA assembly, multiple CRISPR vectors can be constructed in parallel in a single cloning reaction, making the construction of large numbers of CRISPR vectors a simple task. Tomato hairy roots are an excellent model system to validate CRISPR vectors and generate mutant materials.

토마토 털이 뿌리의 생성에 의한 높은 처리량 CRISPR 벡터 건설 및 DNA 수정의 특성

Targeted DNA mutations generated by vectors with clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 technology have proven useful for ...

Hybrid-Cut: An Improved Sectioning Method for Recalcitrant Plant Tissue Samples

Maintaining plant section integrity is essential for studying detailed anatomical structures at the cellular, tissue, or even organ level. However, some plant cells have rigid cell walls, tough fibers and crystals (calcium oxalate, silica, etc.), and high water content that often disrupt tissue integrity during plant tissue sectioning.
This study establishes a simple Hybrid-Cut tissue sectioning method. This protocol modifies a paraffin-based sectioning technique and improves the integrity of tissue sections from different plants. Plant tissues were embedded in paraffin before sectioning in a cryostat at -16 &#176;C. Sectioning under low temperature hardened the paraffin blocks, reduced tearing and scratching, and improved tissue integrity significantly. This protocol was successfully applied to calcium oxalate-rich Phalaenopsis orchid tissues as well as recalcitrant tissues such as reproductive organs and leaves of rice, maize, and wheat. In addition, the high quality of tissue sections from Hybrid-Cut could be used in combination with in situ hybridization (ISH) to provide spatial expression patterns of genes of interest. In conclusion, this protocol is particularly useful for recalcitrant plant tissue containing high crystal or silica content. Good quality tissue sections enable morphological and other biological studies.

This protocol describes a simple Hybrid-Cut tissue sectioning method that is useful for recalcitrant plant tissues. Good quality tissue sections enable anatomical studies and other biological studies including in situ hybridization (ISH).

하이브리드 컷 : 난 분해성 식물 조직 샘플에 대한 개선 단면 방법

Maintaining plant section integrity is essential for studying detailed anatomical structures at the cellular, tissue, or even organ level. However, some ...

Activating Autophagy by Aerobic Exercise in Mice

Autophagy is a lysosomal degradation pathway essential for cell homeostasis, function and differentiation. Under stress conditions, autophagy is induced and targets various cargos, such as bulk cytosol, damaged organelles and misfolded proteins, for degradation in lysosomes. Resulting nutrient molecules are recycled back to the cytosol for new protein synthesis and ATP production. Upregulation of autophagy has beneficial effects against the pathogenesis of many diseases, and pharmacological and physiological strategies to activate autophagy have been reported. Aerobic exercise is recently identified as an efficient autophagy inducer in multiple organs in mice, including muscle, liver, heart and brain. Here we show procedures to induce autophagy in vivo by either forced treadmill exercise or voluntary wheel running. We also demonstrate microscopic and biochemical methods to quantitatively analyze autophagy levels in mouse tissues, using the marker proteins LC3 and p62 that are transported to and degraded in lysosomes along with autophagosomes.

Autophagy activation is beneficial in the prevention of a number of diseases. One of the physiological approaches to induce autophagy in vivo is physical exercise. Here we show how to activate autophagy by aerobic exercise and measure autophagy levels in mice.

마우스에 유산소 운동에 의해 자식 작용을 활성화

Autophagy is a lysosomal degradation pathway essential for cell homeostasis, function and differentiation. Under stress conditions, autophagy is induced ...

Laser-Capture Microdissection RNA-Sequencing for Spatial and Temporal Tissue-Specific Gene Expression Analysis in Plants

The development of a complex multicellular organism is governed by distinct cell types that have different transcriptional profiles. To identify transcriptional regulatory networks that govern developmental processes it is necessary to measure the spatial and temporal gene expression profiles of these individual cell types. Therefore, insight into the spatio-temporal control of gene expression is essential to gain understanding of how biological and developmental processes are regulated. Here, we describe a laser-capture microdissection (LCM) method to isolate small number of cells from three barley embryo organs over a time-course during germination followed by transcript profiling. The method consists of tissue fixation, tissue processing, paraffin embedding, sectioning, LCM and RNA extraction followed by real-time PCR or RNA-seq. This method has enabled us to obtain spatial and temporal profiles of seed organ transcriptomes from varying numbers of cells (tens to hundreds), providing much greater tissue-specificity than typical bulk-tissue analyses. From these data we were able to define and compare transcriptional regulatory networks as well as predict candidate regulatory transcription factors for individual tissues. The method should be applicable to other plant tissues with minimal optimization.

Presented here is a protocol for laser-capture microdissection (LCM) of plant tissues. LCM is a microscopic technique for isolating areas of tissue in a contamination-free manner. The procedure includes tissue fixation, paraffin embedding, sectioning, LCM and RNA extraction. RNA is used in the downstream tissue-specific, temporally resolved analysis of transcriptomes.

레이저 캡처 미세 절 RNA-시퀀싱 용 공간 및 측두 조직 -특정 유전자 발현 분석

The development of a complex multicellular organism is governed by distinct cell types that have different transcriptional profiles. To identify ...

Determination of Tripartite Interaction between Two Monomers of a MADS-box Transcription Factor and a Calcium Sensor Protein by BiFC-FRET-FLIM Assay

Protein-protein interactions are an integral part of all biological processes in the cells as they play a crucial role in regulating, maintaining, and amending cellular functions. These interactions are involved in a wide range of phenomena such as signal transduction, pathogen response, cell-cell interactions, metabolic and developmental processes. In the case of transcription factors, these interactions may lead to oligomerization of subunits, sequestering in specific subcellular contexts such as the nucleus, cytoplasm, etc., which, in turn, might have a more profound effect on the expression of the downstream genes. Here, we demonstrate a methodology to visualize in vivo tripartite interaction using Bimolecular Fluorescence Complementation (BiFC) based F&#246;rster Resonance Energy Transfer (FRET) involving Fluorescence Lifetime Imaging (FLIM). Two of the proteins selected for this demonstration interact as BiFC partners, and their reconstituted fluorescence activity is used to assay FRET-FLIM with the third partner. Four to five-week-old growth-chamber-grown Nicotiana benthamiana plants have been used as the model plant system for this demonstration.

Here we present, a method to visualize ternary complex formation between three protein partners using fluorescent-tagged proteins by BiFC based FRET-FLIM assay. This method is valuable for studying protein-protein interaction complexes in vivo.

BIFC-FRET-FLIM 분석체에 의한 MADS 박스 전사 계수의 두 단백제와 칼슘 센서 단백질 간의 삼자 상호 작용 결정

Protein-protein interactions are an integral part of all biological processes in the cells as they play a crucial role in regulating, maintaining, and ...

Comparison of Methods for Isolating Entomopathogenic Fungi from Soil Samples

The goal of the present study is to compare the effectiveness of using insect baits versus artificial selective medium for isolating entomopathogenic fungi (EPF) from soil samples. The soil is a rich habitat for microorganisms, including EPF particularly belonging to the genera Metarhizium and Beauveria, which can regulate arthropod pests. Biological products based on fungi are available in the market mainly for agricultural arthropod pest control. Nevertheless, despite the high endemic biodiversity, only a few strains are used in commercial bioproducts worldwide. In the present study, 524 soil samples were cultured on potato dextrose agar enriched with yeast extract supplemented with chloramphenicol, thiabendazole, and cycloheximide (CTC medium). The growth of fungal colonies was observed for 3 weeks. All Metarhizium and Beauveria EPF were morphologically identified at the genus level. Additionally, some isolates were molecularly identified at the species level. Twenty-four out of these 524 soil samples were also surveyed for EPF occurrence using the insect bait method (Galleria mellonella and Tenebrio molitor). A total of 51 EPF strains were isolated (41 Metarhizium spp. and 10 Beauveria spp.) from the 524 soil samples. All fungal strains were isolated either from croplands or grasslands. Of the 24 samples selected for comparison, 91.7% were positive for EPF using Galleria bait, 62.5% using Tenebrio bait, and 41.7% using CTC. Our results suggested that using insect baits to isolate the EPF from the soil is more efficient than using the CTC medium. The comparison of isolation methods in addition to the identification and conservation of EPF has a positive impact on the knowledge about biodiversity. The improvement of EPF collection supports scientific development and technological innovation.

Entomopathogenic fungal colonies are isolated from tropical soil samples using Tenebrio bait, Galleria bait, as well as selective artificial medium, i.e., potato dextrose agar enriched with yeast extract supplemented with chloramphenicol, thiabendazole, and cycloheximide (CTC medium).

토양 샘플에서 엔토모병원성 곰팡이를 분리하는 방법의 비교

The goal of the present study is to compare the effectiveness of using insect baits versus artificial selective medium for isolating entomopathogenic ...