This work benefited from the networking activities coordinated within the EU-funded COST ACTION FA1106 "An integrated systems approach to determine the developmental mechanisms controlling fleshy fruit quality in tomato and grapevine". This work was supported by the 'Completamento del Centro di Genomica Funzionale Vegetale' project funded by the CARIVERONA Bank Foundation and by the 'Valorizzazione dei Principali Vitigni Autoctoni Italiani e dei loro Terroir (Vigneto)' project funded by the Italian Ministry of Agricultural and Forestry Policies. SDS was financed by the Italian Ministry of University and Research FIRB RBFR13GHC5 project "The Epigenomic Plasticity of Grapevine in Genotype per Environment Interactions".

<ol>
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The authors have nothing to disclose.

이 문서에서는 포도 베리 떼루아 개념을 해석하는 데 사용되는 대사 체학, transcriptomics 및 통계 분석 프로토콜을 설명합니다. HPLC-ESI-MS에 의해 대사 체학 분석 동시에 대사 산물을 대량을 검출하기에 충분히 민감하지만, 상대적 정량은 매트릭스 효과 이온 억제 / 강화에 의해 영향을 받는다. 그러나 유사한 접근 방식은 이미 조기는 열매의 숙성 및 수확 후 고사를 설명하는 데 사용되었으며, 매트릭...

식물 게놈 전 사체, 프로테옴 및 metabolomes에 기초하여 대규모 데이터 분석은 포도 식물과 그 환경 사이의 상호 작용을 반영하는 와인 떼루아 특성과 같은 복잡한 시스템의 작동에 대한 새로운 통찰력을 제공한다. 동일한 포도 나무의 클론이 다른 포도원에서 재배하는 경우에도 와인의 떼루아 구별 할 수 있기 때문에 클론 게놈이 동일하기 때문에, 게놈 분석은 거의 사용이다. 대신 유전자 발현 와인의 품질 특성을 결정하는 열매의 대사 특성 간의 상관 관계를 볼 필요가있다. 마이크로 어레이에 고정 된 프로브와 같은 하이브리드와 같은 보편적 인 특성을 이용하여 정량 분석을 용이하게 모든 성적 증명서의 유사한 화학적 성질,의 사체 혜택의 수준에서 유전자 발현의 분석. 프로테오믹스 A의 대조적으로, 보편적 인 분석 방법차 대사 체학 때문에 개별 단백질 및 대사 산물의 거대한 물리적, 화학적 다양성을 더 도전입니다. 대사 체학의 경우 개별 대사 산물의 크기, 극성, 풍부하고 변동성에 크게 다르기 때문에 이러한 다양성은 훨씬 더 극단적이다, 그래서 하나의 추출 과정이나 분석 방법 론적 접근 방식을 제공합니다. 비 휘발성 대사 적합한 분석 플랫폼 중, 고성능 액체 크로마토 그래피에 기초하여 그 질량 분석계에 연결된 (HPLC-MS)는 훨씬 더 민감 자외선 또는 다이오드 배열 검출기를 가진 HPLC (HPLC-UV, HPLC-DAD 같은 대안보다 ) 또는 HPLC-MS에 의해 핵 자기 공명 (NMR) 분광법 있지만 정량 분석은 이러한 매트릭스 효과 이온 진압 / 인핸스 1-3 현상에 의해 영향을받을 수있다. (HPLC-E를 전기 분무 이온화 소스를 사용하여 HPLC-MS에 의한 조기는 포도 열매의 분석시 이러한 영향의 조사SI-MS)은 가장 낮은 체류 시간과 당 및 다른 분자 강하게 아마도이 영역에서 분자의 다수를 반영 과소 것으로 나타났다 및 다른 분자의 존재 량 매트릭스 효과에 의해 과대 또는받지 과소 평가 될 수 있다는 하지만, 매트릭스 효과에 대한 데이터 정규화는 전체 결과 4,5에 제한된 영향을 미칠 것 같았다. 본원에 기재된 방법은 숙성 중에 포도 열매에서 높은 수준으로 축적 매체 극성 대사 산물의 분석을 위해 최적화되고, 이는 상당히 떼루아의 영향. 그들은 함께 색상, 맛과 와인의 건강 관련 특성을 결정 안토시아닌, 플라 보놀, flavan-3-OLS, 프로시아니딘, 다른 플라보노이드, 레스베라트롤, 스틸 벤, 히드 록시 산, 하이드 록시 산을 포함한다. HPLC-MS에 의한 정량은 신뢰할 수 있기 때문에 같은 당 및 지방족 유기산과 같은 다른 대사는, 때문에 effec 행렬에 무시됩니다t 이온 억제 현상 (5). 이 방법에 의해 선택된 극성의 범위 내에서 접근 가능 6만큼 다른 대사 물질을 검출하는 것을 목적으로 불특정이다. 포도 나무의 성적 증명서의 수천을 동시에 모니터링 할 수 Transcriptomics 방법은 전체 포도 게놈 서열 7,8의 가용성에 의해 촉진된다. 높은 처리량의 cDNA 서열에 기초하여 조기 transcriptomics 방법 일괄 RNA-SEQ 기술로 설명한 절차의 집합으로 차세대 서열의 출현으로 발전해왔다. 이 방법은 빠르게 transcriptomics 연구에 대한 선택의 방법이되고있다. 그러나, 성적 증명서의 수천 하이브리드에 의해 병렬로 정량화 할 수 있도록 마이크로 어레이를 기반으로 문학의 큰 몸은 포도 나무에 대한 축적하고있다. RNA-서열이 주류 기술이되기 전에 실제로, 많은 전용 상용 마이크로 어레이 플랫폼은 있었다개발 허용 포도 나무의 사체는 훌륭한 세부 사항에 검사합니다. 플랫폼의 광대 한 다양한 가운데, 두 개의 게놈 전체의 전 사체 분석 (9)을 허용했다. 가장 진화 배열 따라서 각 실험의 비용을 절감하는 하나의 장치에서 최대 12 개의 독립적 인 샘플의 혼성화시켰다. 12 서브 어레이는 각각 29,549 포도 나무의 성적을 나타내는 135,000 60 머 프로브를 구성. 이 장치는 시험 10-24의 다수에 사용되고있다. 이 두 플랫폼은 이제 중단되었지만 새 사용자 정의 마이크로 어레이는 최근 설계되고있어 추가 새로 발견 된 포도 나무의 유전자 (25)를 나타내는 프로브의 더 큰 수를 포함로 최근 개발을 나타냅니다. transcriptomics와 대사 체학 분석에 의해 생성 된 대형 판매 데이터 세트는 다른 형태 사이의 상관 관계를 결정하기 위해 다변량 기법을 포함한 데이터 분석에 적합한 통계 방법을 필요로데이터의. 가장 널리 사용되는 변수 기술들은 투영에 기초하여 해당하며, 여기에는 주성분 분석 (PCA), 또는, 자율 될 수있는 잠재적 인 구조 판별 분석 (O2PLS-DA) (26)에 양방향 정사영 같은 감독. 이 문서에 제시된 프로토콜은 샘플 그룹 간의 차이를 식별하기 위해 탐색 적 데이터 분석 및 O2PLS-DA에 대한 PCA를 사용합니다.

<table><tbody><tr><td>Mill Grinder</td><td>IKA</td><td>IKA A11 basic</td><td></td></tr><tr><td>HPLC Autosampler</td><td>Beckman Coulter</td><td>&amp;nbsp;-</td><td>System Gold 508 Autosampler</td></tr><tr><td>HPLC System</td><td>Beckman Coulter</td><td>&amp;nbsp;-</td><td>System Gold 127 Solvent Module HPLC</td></tr><tr><td>C18 Guard Column</td><td>Grace</td><td>&amp;nbsp;-</td><td>Alltima HP C18 (7.5 mm x 2.1 mm; 5 &amp;mu;m) Guard Column</td></tr><tr><td>C18 Column</td><td>Grace</td><td>&amp;nbsp;-</td><td>Alltima HP C18 (150 mm x 2.1 mm; 3 &amp;mu;m) Column</td></tr><tr><td>Mass Spectometer</td><td>Bruker Daltonics</td><td>&amp;nbsp;-</td><td>Bruker Esquire 6000; The mass spectometer was equipped with an ESI source and the analyzer was an ion trap.</td></tr><tr><td>Extraction solvents and HPLC buffers</td><td>Sigma</td><td>34966</td><td>Methanol LC-MS grade</td></tr><tr><td>Sigma</td><td>94318</td><td>Formic acid LC-MS grade</td><td></td></tr><tr><td>Sigma</td><td>34967</td><td>Acetonitrile LC-MS grade</td><td></td></tr><tr><td>Sigma</td><td>39253</td><td>Water&amp;nbsp; LC-MS grade</td><td></td></tr><tr><td>Minisart RC 4 Syringe filters (0.2 &amp;mu;m)</td><td>Sartorius</td><td>17764</td><td></td></tr><tr><td>Softwares for data collection (a) and processing (b)</td><td>Bruker Daltonics</td><td>-</td><td>Bruker Daltonics Esquire 5.2 Control (a); Esquire 3.2 Data Analysis and MzMine 2.2 softwares (b)</td></tr><tr><td>Spectrum Plant Total RNA kit</td><td>Sigma-Aldrich</td><td>STRN250-1KT</td><td>For total RNA extractino from grape pericarps</td></tr><tr><td>Nanodrop 1000</td><td>Thermo Scientific</td><td>1000</td><td></td></tr><tr><td>BioAnalyzer 2100</td><td>Agilent Technologies</td><td>G2939A</td><td></td></tr><tr><td>RNA 6000 Nano Reagents</td><td>Agilent Technologies</td><td>5067-1511</td><td></td></tr><tr><td>RNA Chips</td><td>Agilent Technologies</td><td>5067-1511</td><td></td></tr><tr><td>Agilent Gene Expression Wash Buffer 1</td><td>Agilent Technologies</td><td>5188-5325</td><td></td></tr><tr><td>Agilent Gene Expression Wash Buffer 2</td><td>Agilent Technologies</td><td>5188-5326</td><td></td></tr><tr><td>LowInput QuickAmp Labeling kit One-Color</td><td>Agilent Technologies</td><td>5190-2305</td><td></td></tr><tr><td>Kit RNA Spike In - One-Color</td><td>Agilent Technologies</td><td>5188-5282</td><td></td></tr><tr><td>Gene Expression Hybridization Kit</td><td>Agilent Technologies</td><td>5188-5242</td><td></td></tr><tr><td>RNeasy Mini Kit (50)</td><td>Qiagen</td><td>74104</td><td>For cRNA Purification</td></tr><tr><td>Agilent SurePrint HD 4X44K 60-mer Microarray</td><td>Agilent Technologies</td><td>G2514F-048771&amp;nbsp;</td><td></td></tr><tr><td>eArray</td><td>Agilent Technologies</td><td>-</td><td>&lt;a href=&quot;https://earray.chem.agilent.com/earray/&quot;&gt;https://earray.chem.agilent.com/earray/&lt;/a&gt;</td></tr><tr><td>Gasket slides</td><td>Agilent Technologies</td><td>G2534-60012</td><td>Enable Agilent SurePrint Microarray 4-array Hybridization</td></tr><tr><td>Thermostatic bath</td><td>Julabo</td><td>-</td><td></td></tr><tr><td>Hybridization Chamber</td><td>Agilent Technologies</td><td>G2534-60001</td><td></td></tr><tr><td>Microarray Hybridization Oven</td><td>Agilent Technologies</td><td>G2545A</td><td></td></tr><tr><td>Hybridization Oven Rotator Rack</td><td>Agilent Technologies</td><td>G2530-60029</td><td></td></tr><tr><td>Rotator Rack Conversion Rod</td><td>Agilent Technologies</td><td>G2530-60030</td><td></td></tr><tr><td>Staining kit</td><td>Bio-Optica</td><td>10-2000</td><td>Slide-staining dish and Slide rack</td></tr><tr><td>Magnetic stirrer device</td><td>AREX Heating Magnetic Stirrer</td><td>F20540163&amp;nbsp;</td><td></td></tr><tr><td>Thermostatic Oven</td><td>Thermo Scientific</td><td>Heraeus - 6030</td><td></td></tr><tr><td>Agilent Microarray Scanner</td><td>Agilent Technologies</td><td>G2565CA</td><td></td></tr><tr><td>Scanner Carousel, 48-position</td><td>Agilent Technologies</td><td>G2505-60502</td><td></td></tr><tr><td>Slide Holders</td><td>Agilent Technologies</td><td>G2505-60525</td><td></td></tr><tr><td>Feature extraction software v11.5</td><td>Agilent Technologies</td><td>-</td><td>inside the Agilent Microarray Scanner G2565CA</td></tr><tr><td>SIMCA + V13 Software</td><td>Umetrics</td><td></td><td></td></tr></tbody></table>

the terroir concept interpreted through grape berry metabolomics and transcriptomics

떼루아 특히 서식지 및 관리 방법에 따라 같은 포도 (유럽 종 포도)로 작물의 특성에 영향을 미치는 환경 요인의 조합을 의미합니다. 이 문서에서는 특정 떼루아 서명이 다변량 통계 분석을 사용하여 포도의 품종, 조기의 베리 대사 체 및 사체에서 검출 할 수있는 방법을 보여줍니다. 이 방법은 먼저 적절한 샘플링 계획이 필요합니다. 이 사례에서, 조기 품종의 특정 클론 유전 적 차이를 최소화하도록 선택되고, 샘플은 세 가지 성장 계절 동안 세 가지 매크로 영역을 나타내는 일곱 포도에서 수집 하였다. 타겟팅되지 않은 LC-MS의 대사 체학 접근 MZmine 소프트웨어 및 단편화 트리 분석을 기초 대사 식별 전략을 사용하여 효율적으로 데이터 처리를 수반 높은 민감도 때문에 권장된다. 종합 사체 분석은 마이크로 어레이를 사용하여 달성 될 수있다함유 프로브 다른 TERROIRS의 컨텍스트에서 모든 차등 유전자 발현의 동시 분석을 가능 ~ 모든 예측 리세 유전자의 99 %를 커버. 마지막으로, 투영 방법에 기초하여 다변량 데이터 분석은 대사 체학 및 transcriptomics 데이터 통합 ​​정보의 상관 관계를 식별하기 위해 상세하게 분석 될 수 있도록 강력한 빈티지 특정 효과를 극복하기 위해 사용될 수있다.

이 문서에서 설명하는 사례 연구는 상대적으로 189 샘플 중 정량 분자 이온 플러스 자신의 동위 원소, 부가 물 및 일부 조각을 포함하여 552 신호 (m / Z 기능), (7 포도원 × 3 숙성 단계 × 3 성장 계절 X를 포함하는 최종 데이터 매트릭스를 산출 3 생물 복제). 데이터 포인트의 총 수는 따라서 104,328이었다. 분할 트리 분석은 대사 플러스 부가 물, 동위 원소 및 조각에 해당?...

Watch this Scientific Journal Video about The Terroir Concept Interpreted through Grape Berry Metabolomics and Transcriptomics at JoVE.com

The Terroir Concept Interpreted through Grape Berry Metabolomics and Transcriptomics

이 문서는 즉 떼루아 개념에 대한 통찰력을 얻기 위해 포도 베리 성적 증명서 및 대사에 타겟이 불분명 한 대사 체학, transcriptomics 및 다변량 통계 분석의 응용 프로그램을 설명 베리 품질 특성에 대한 환경의 영향.

포도 베리 대사 체학 및 Transcriptomics을 통해 해석 떼루아 개념

Terroir refers to the combination of environmental factors that affect the characteristics of crops such as grapevine (Vitis vinifera) according to ...

the-terroir-concept-interpreted-through-grape-berry-metabolomics

Universidad San Sebastian

Research

JoVE Journal

Genetics

10.3K 조회수.  University of Verona.  이 문서는 즉 떼루아 개념에 대한 통찰력을 얻기 위해 포도 베리 성적 증명서 및 대사에 타겟이 불분명 한 대사 체학, transcriptomics 및 다변량 통계 분석의 응용 프로그램을 설명 베리 품질 특성에 대한 환경의 영향. 

비디오: The Terroir Concept Interpreted through Grape Berry Metabolomics and Transcriptomics

In Vitro Transcription Assays and Their Application in Drug Discovery

In vitro transcription assays have been developed and widely used for many years to study the molecular mechanisms involved in transcription. This process requires multi-subunit DNA-dependent RNA polymerase (RNAP) and a series of transcription factors that act to modulate the activity of RNAP during gene expression. Sequencing gel electrophoresis of radiolabeled transcripts is used to provide detailed mechanistic information on how transcription proceeds and what parameters can affect it. In this paper we describe the protocol to study how the essential elongation factor NusA regulates transcriptional pausing, as well as a method to identify an antibacterial agent targeting transcription initiation through inhibition of RNAP holoenzyme formation. These methods can be used a as platform for the development of additional approaches to explore the mechanism of action of the transcription factors which still remain unclear, as well as new antibacterial agents targeting transcription which is an underutilized drug target in antibiotic research and development.

In Vitro Transcription Assays and Their Application in Drug Discovery

In this manuscript, we describe a protocol to functionally examine transcription and the inhibitory activity of antibacterial agents targeting bacterial transcription.

시험 관내 전사 분석 실험 및 약물 발견에의 응용

In vitro transcription assays have been developed and widely used for many years to study the molecular mechanisms involved in transcription. This process ...

연구

유전학

Delivery of Nucleic Acids through Embryo Microinjection in the Worldwide Agricultural Pest Insect, Ceratitis capitata

The Mediterranean fruit fly (medfly) Ceratitis capitata (Wiedemann) (Diptera: Tephritidae) is a pest species with extremely high agricultural relevance. This is due to its reproductive behavior: females damage the external surface of fruits and vegetables when they lay eggs and the hatched larvae feed on their pulp. Wild C. capitata populations are traditionally controlled through insecticide spraying and/or eco-friendly approaches, the most successful being the Sterile Insect Technique (SIT). The SIT relies on mass-rearing, radiation-based sterilization and field release of males that retain their capacity to mate but are not able to generate fertile progeny. The advent and the subsequent rapid development of biotechnological tools, together with the availability of the medfly genome sequence, has greatly boosted our understanding of the biology of this species. This favored the proliferation of new strategies for genome manipulation, which can be applied to population control.
In this context, embryo microinjection plays a dual role in expanding the toolbox for medfly control. The ability to interfere with the function of genes that regulate key biological processes, indeed, expands our understanding of the molecular machinery underlying medfly invasiveness. Furthermore, the ability to achieve germ-line transformation facilitates the production of multiple transgenic strains that can be tested for future field applications in novel SIT settings. Indeed, genetic manipulation can be used to confer desirable traits that can, for example, be used to monitor sterile male performance in the field, or that can result in early life-stage lethality. Here we describe a method to microinject nucleic acids into medfly embryos to achieve these two main goals.

Delivery of Nucleic Acids through Embryo Microinjection in the Worldwide Agricultural Pest Insect, Ceratitis capitata

The Mediterranean fruit fly (medfly) Ceratitis capitata (Diptera: Tephritidae) is a worldwide pest of agriculture. A deeper understanding of its biology is key to control medfly populations and thus reduce economic impact. Embryo microinjection is a fundamental tool allowing both germ-line transformation and reverse genetics studies in this species.

세계적인 농업 해충 곤충의 배아 미세 주입을 통해 핵산 배달,<em&gt; Ceratitis의 capitata</em

The Mediterranean fruit fly (medfly) Ceratitis capitata (Wiedemann) (Diptera: Tephritidae) is a pest species with extremely high agricultural relevance. ...

Leveraging CyVerse Resources for De Novo Comparative Transcriptomics of Underserved (Non-model) Organisms

This workflow allows novice researchers to leverage advanced computational resources such as cloud computing to carry out pairwise comparative transcriptomics. It also serves as a primer for biologists to develop data scientist computational skills, e.g. executing bash commands, visualization and management of large data sets. All command line code and further explanations of each command or step can be found on the wiki (<a href="https://wiki.cyverse.org/wiki/x/dgGtAQ">https://wiki.cyverse.org/wiki/x/dgGtAQ</a>). The Discovery Environment and Atmosphere platforms are connected together through the CyVerse Data Store. As such, once the initial raw sequencing data has been uploaded there is no more need to transfer large data files over an Internet connection, minimizing the amount of time needed to conduct analyses. This protocol is designed to analyze only two experimental treatments or conditions. Differential gene expression analysis is conducted through pairwise comparisons, and will not be suitable to test multiple factors. This workflow is also designed to be manual rather than automated. Each step must be executed and investigated by the user, yielding a better understanding of data and analytical outputs, and therefore better results for the user. Once complete, this protocol will yield de novo assembled transcriptome(s) for underserved (non-model) organisms without the need to map to previously assembled reference genomes (which are usually not available in underserved organism). These de novo transcriptomes are further used in pairwise differential gene expression analysis to investigate genes differing between two experimental conditions. Differentially expressed genes are then functionally annotated to understand the genetic response organisms have to experimental conditions. In total, the data derived from this protocol is used to test hypotheses about biological responses of underserved organisms.

Leveraging CyVerse Resources for De Novo Comparative Transcriptomics of Underserved Non-model Organisms

This protocol outlines a comparative de novo transcriptome assembly and annotation workflow for novice bioinformaticians. The workflow is available for free entirely through CyVerse and connected by the Data Store. Command line and graphical user interfaces are used, but all code needed is available to copy and paste.

CyVerse 리소스 활용<em&gt; 드 노보</em&gt; 비포장 (비 모델) 생물의 비교 전이학

This workflow allows novice researchers to leverage advanced computational resources such as cloud computing to carry out pairwise comparative ...

Controllable Ion Channel Expression through Inducible Transient Transfection

Transfection, the delivery of foreign nucleic acids into a cell, is a powerful tool in protein research. Through this method, ion channels can be investigated through electrophysiological analysis, biochemical characterization, mutational studies, and their effects on cellular processes. Transient transfections offer a simple protocol in which the protein becomes available for analysis within a few hours to days. Although this method presents a relatively straightforward and time efficient protocol, one of the critical components is calibrating the expression of the gene of interest to physiological relevant levels or levels that are suitable for analysis. To this end, many different approaches that offer the ability to control the expression of the gene of interest have emerged. Several stable cell transfection protocols provide a way to permanently introduce a gene of interest into the cellular genome under the regulation of a tetracycline-controlled transcriptional activation. While this technique produces reliable expression levels, each gene of interest requires a few weeks of skilled work including calibration of a killing curve, selection of cell colonies, and overall more resources. Here we present a protocol that uses transient transfection of the Transient Receptor Potential cation channel subfamily V member 1 (TRPV1) gene in an inducible system as an efficient way to express a protein in a controlled manner which is essential in ion channel analysis. We demonstrate that using this technique, we are able to perform calcium imaging, whole cell, and single channel analysis with controlled channel levels required for each type of data collection with a single transfection. Overall, this provides a replicable technique that can be used to study ion channels structure and function.

Studying ion channels through a heterologously expressing system has become a core technique in biomedical research. In this manuscript, we present a time efficient method to achieve tightly controlled ion channel expression by performing transient transfection under the control of an inducible promoter.

유도 성 과도 형질을 통해 제어 이온 채널 식

Transfection, the delivery of foreign nucleic acids into a cell, is a powerful tool in protein research. Through this method, ion channels can be ...

Transcriptomic Analysis of C. elegans RNA Sequencing Data Through the Tuxedo Suite on the Galaxy Project

Next generation sequencing (NGS) technologies have revolutionized the nature of biological investigation. Of these, RNA Sequencing (RNA-Seq) has emerged as a powerful tool for gene-expression analysis and transcriptome mapping. However, handling RNA-Seq datasets requires sophisticated computational expertise and poses inherent challenges for biology researchers. This bottleneck has been mitigated by the open access Galaxy project that allows users without bioinformatics skills to analyze RNA-Seq data, and the Database for Annotation, Visualization, and Integrated Discovery (DAVID), a Gene Ontology (GO) term analysis suite that helps derive biological meaning from large data sets. However, for first-time users and bioinformatics' amateurs, self-learning and familiarization with these platforms can be time-consuming and daunting. We describe a straightforward workflow that will help C. elegans researchers to isolate worm RNA, conduct an RNA-Seq experiment and analyze the data using Galaxy and DAVID platforms. This protocol provides stepwise instructions for using the various Galaxy modules for accessing raw NGS data, quality-control checks, alignment, and differential gene expression analysis, guiding the user with parameters at every step to generate a gene list that can be screened for enrichment of gene classes or biological processes using DAVID. Overall, we anticipate that this article will provide information to C. elegans researchers undertaking RNA-Seq experiments for the first time as well as frequent users running a small number of samples.

Transcriptomic Analysis of C. elegans RNA Sequencing Data Through the Tuxedo Suite on the Galaxy Project

Galaxy and DAVID have emerged as popular tools that allow investigators without bioinformatics training to analyze and interpret RNA-Seq data. We describe a protocol for C. elegans researchers to perform RNA-Seq experiments, access and process the dataset using Galaxy and obtain meaningful biological information from the gene lists using DAVID.

의 Transcriptomic 분석<em&gt; C</em&gt;.<em&gt; 간스</em갤럭시 프로젝트에 턱시도 제품군을 통해&gt; RNA 시퀀싱 데이터

Next generation sequencing (NGS) technologies have revolutionized the nature of biological investigation. Of these, RNA Sequencing (RNA-Seq) has emerged ...

Untargeted Liquid Chromatography-Mass Spectrometry-Based Metabolomics Analysis of Wheat Grain

Understanding the interactions between genes, the environment and management in agricultural practice could allow more accurate prediction and management of product yield and quality. Metabolomics data provides a read-out of these interactions at a given moment in time and is informative of an organism&#39;s biochemical status. Further, individual metabolites or panels of metabolites can be used as precise biomarkers for yield and quality prediction and management. The plant metabolome is predicted to contain thousands of small molecules with varied physicochemical properties that provide an opportunity for a biochemical insight into physiological traits and biomarker discovery. To exploit this, a key aim for metabolomics researchers is to capture as much of the physicochemical diversity as possible within a single analysis. Here we present a liquid chromatography-mass spectrometry-based untargeted metabolomics method for the analysis of field-grown wheat grain. The method uses the liquid chromatograph quaternary solvent manager to introduce a third mobile phase and combines a traditional reversed-phase gradient with a lipid-amenable gradient. Grain preparation, metabolite extraction, instrumental analysis and data processing workflows are described in detail. Good mass accuracy and signal reproducibility were observed, and the method yielded approximately 500 biologically relevant features per ionization mode. Further, significantly different metabolite and lipid feature signals between wheat varieties were determined.

A method for the untargeted analysis of wheat grain metabolites and lipids is presented. The protocol includes an acetonitrile metabolite extraction method and reversed phase liquid chromatography-mass spectrometry methodology, with acquisition in positive and negative electrospray ionization modes.

밀 곡물의 비표적 액체 크로마토그래피 질량 분광계 기반 대사체학 분석

Understanding the interactions between genes, the environment and management in agricultural practice could allow more accurate prediction and management ...

생화학

Profiling Volatile Compounds in Blackcurrant Fruit using Headspace Solid-Phase Microextraction Coupled to Gas Chromatography-Mass Spectrometry

There is an increasing interest in measuring volatile organic compounds (VOCs) emitted by ripe fruits for the purpose of breeding varieties or cultivars with enhanced organoleptic characteristics and thus, to increase consumer acceptance. High-throughput metabolomic platforms have been recently developed to quantify a wide range of metabolites in different plant tissues, including key compounds responsible for fruit taste and aroma quality (volatilomics). A method using headspace solid-phase microextraction (HS-SPME) coupled with gas chromatography-mass spectrometry (GC-MS) is described here for the identification and quantification of VOCs emitted by ripe blackcurrant fruits, a berry highly appreciated for its flavor and health benefits.
Ripe fruits of blackcurrant plants (Ribes nigrum) were harvested and directly frozen in liquid nitrogen. After tissue homogenization to produce a fine powder, samples were thawed and immediately mixed with sodium chloride solution. Following centrifugation, the supernatant was transferred into a headspace glass vial containing sodium chloride. VOCs were then extracted using a solid-phase microextraction (SPME) fiber and a gas chromatograph coupled to an ion trap mass spectrometer. Volatile quantification was performed on the resulting ion chromatograms by integrating peak area, using a specific m/z ion for each VOC. Correct VOC annotation was confirmed by comparing retention times and mass spectra of pure commercial standards run under the same conditions as the samples. More than 60 VOCs were identified in ripe blackcurrant fruits grown in contrasting European locations. Among the identified VOCs, key aroma compounds, such as terpenoids and C6 volatiles, can be used as biomarkers for blackcurrant fruit quality. In addition, advantages and disadvantages of the method are discussed, including prospective improvements. Furthermore, the use of controls for batch correction and minimization of drift intensity have been emphasized.

A headspace solid-phase microextraction-gas-chromatography platform is described here for fast, reliable, and semi-automated volatile identification and quantification in ripe blackcurrant fruits. This technique can be used to increase knowledge about fruit aroma and to select cultivars with enhanced flavor for the purpose of breeding.

헤드스페이스 고체-위상 미세 추출을 사용하여 블랙커런트 과일의 휘발성 화합물 프로파일링

There is an increasing interest in measuring volatile organic compounds (VOCs) emitted by ripe fruits for the purpose of breeding varieties or cultivars ...

Annotation of Plant Gene Function via Combined Genomics, Metabolomics and Informatics

Given the ever expanding number of model plant species for which complete genome sequences are available and the abundance of bio-resources such as knockout mutants, wild accessions and advanced breeding populations, there is a rising burden for gene functional annotation. In this protocol, annotation of plant gene function using combined co-expression gene analysis, metabolomics and informatics is provided (Figure 1). This approach is based on the theory of using target genes of known function to allow the identification of non-annotated genes likely to be involved in a certain metabolic process, with the identification of target compounds via metabolomics. Strategies are put forward for applying this information on populations generated by both forward and reverse genetics approaches in spite of none of these are effortless. By corollary this approach can also be used as an approach to characterise unknown peaks representing new or specific secondary metabolites in the limited tissues, plant species or stress treatment, which is currently the important trial to understanding plant metabolism.

Combination of genomics, co-expression gene analysis and the identification of target compounds via metabolism give gene functional annotation.

결합 지노 믹스, Metabolomics와 정보학 통해 식물 유전자 기능의 주석

Given the ever expanding number of model plant species for which complete genome sequences are available and the abundance of bio-resources such as ...

생물학

Comprehensive Workflow for the Genome-wide Identification and Expression Meta-analysis of the ATL E3 Ubiquitin Ligase Gene Family in Grapevine

Classification and nomenclature of genes in a family can significantly contribute to the description of the diversity of encoded proteins and to the prediction of family functions based on several features, such as the presence of sequence motifs or of particular sites for post-translational modification and the expression profile of family members in different conditions. This work describes a detailed protocol for gene family characterization. Here, the procedure is applied to the characterization of the Arabidopsis T&#243;xicos in Levadura (ATL) E3 ubiquitin ligase family in grapevine. The methods include the genome-wide identification of family members, the characterization of gene localization, structure, and duplication, the analysis of conserved protein motifs, the prediction of protein localization and phosphorylation sites as well as gene expression profiling across the family in different datasets. Such procedure, which could be extended to further analyses depending on experimental purposes, could be applied to any gene family in any plant species for which genomic data are available, and it provides valuable information to identify interesting candidates for functional studies, giving insights into the molecular mechanisms of plant adaptation to their environment.

This article describes the procedure for the identification and characterization of a gene family in grapevine applied to the family of Arabidopsis T&#243;xicos in Levadura (ATL) E3 ubiquitin ligases.

게놈 넓은 식별 및 소문의 ATL E3 유비퀴틴 리가 유전자 가족의 식 메타 분석을 위한 포괄적인 워크플로

Classification and nomenclature of genes in a family can significantly contribute to the description of the diversity of encoded proteins and to the ...

Amplicon metagenomics를 사용한 포도의 박테리아 군집 프로파일링

Profiling the Bacterial Community of Fermenting Traminette Grapes during Wine Production using Metagenomic Amplicon Sequencing

Advances in sequencing technology and the relatively easy access to the use of bioinformatics tools to profile microbial community structures have facilitated a better understanding of both culturable and non-culturable microbes in grapes and wine. During industrial fermentation, microbes, known and unknown, are often responsible for product development and off-flavor. Therefore, profiling the bacteria from grape to wine can enable an easy understanding of in situ microbial dynamics. In this study, the bacteria of Traminette grapes must undergoing fermentation, and the final wine were subjected to DNA extraction that yielded 15 ng/&#181;L to 87 ng/&#181;L. The 16S amplicon of the hypervariable region of the V4 region was sequenced, relatively abundant bacteria consisting of phyla Proteobacteria, Actinobacteriota, Firmicutes, Bacteroidota, Fusobacteriota and followed by the Verrucomicrobiota, Halobacterota, Desulfobacterota, Myxococcota, and Acidobacteriota. A Venn diagram analysis of the shared unique operational taxonomic units (OTU) revealed that 15 bacteria phyla were common to both grape must, fermenting stage, and final wine. Phyla that were not previously reported were detected using the 16S amplicon sequencing, as well as genera such as Enterobacteriaceae and Lactobacillaceae. Variation in the organic nutrient use in wine and its impact on bacteria was tested; Traminette R tank containing Fermaid O and Traminette L stimulated with Stimula Sauvignon blanc + Fermaid O. Alpha diversity using the Kruskal-Wallis test determined the degree of evenness. The beta diversity indicated a shift in the bacteria at the fermentation stage for the two treatments, and the final wine bacteria looked similar. The study confirmed that 16S amplicon sequencing can be used to monitor bacteria changes during wine production to support quality and better utilization of grape bacteria during wine production.

저자 스포트라이트: 차세대 염기서열분석(Next-Generation Sequencing)을 통한 발효 마이크로바이옴 탐구

Here, we present a protocol to describe amplicon metagenomic for determining the bacterial community of Traminette grapes, fermenting grapes, and final wine.

포도 베리 대사 체학 및 Transcriptomics을 통해 해석 떼루아 개념

요약

더 많은 비디오 탐색

이 비디오의 챕터

시험 관내 전사 분석 실험 및 약물 발견에의 응용

세계적인 농업 해충 곤충의 배아 미세 주입을 통해 핵산 배달,

CyVerse 리소스 활용

유도 성 과도 형질을 통해 제어 이온 채널 식

의 Transcriptomic 분석

밀 곡물의 비표적 액체 크로마토그래피 질량 분광계 기반 대사체학 분석

헤드스페이스 고체-위상 미세 추출을 사용하여 블랙커런트 과일의 휘발성 화합물 프로파일링

결합 지노 믹스, Metabolomics와 정보학 통해 식물 유전자 기능의 주석

게놈 넓은 식별 및 소문의 ATL E3 유비퀴틴 리가 유전자 가족의 식 메타 분석을 위한 포괄적인 워크플로

메타게놈 앰플리콘 염기서열분석(Metagenomic Amplicon Sequencing)을 사용한 와인 생산 중 발효 트라미네트 포도의 박테리아 군집 프로파일링