Cold Spring Harbor protocols最新文献_第8页

Root Anatomical Imaging and Phenotyping in Maize. 玉米根部解剖成像和表型分析

Cold Spring Harbor protocols Pub Date : 2024-08-30 DOI: 10.1101/pdb.top108454

Jagdeep Singh Sidhu, Hannah M Schneider

{"title":"Root Anatomical Imaging and Phenotyping in Maize.","authors":"Jagdeep Singh Sidhu, Hannah M Schneider","doi":"10.1101/pdb.top108454","DOIUrl":"https://doi.org/10.1101/pdb.top108454","url":null,"abstract":"Root anatomy plays a crucial role in regulating essential processes such as the absorption and movement of water and nutrients in plants. Root anatomy also impacts the energy costs of building and sustaining root tissues, tissue mechanics, and interactions with other organisms. Although several studies in maize have confirmed the functional utility of numerous root anatomical traits, such as that of cortical cell size and number for stress adaptation, there have been significant obstacles in measuring and analyzing root anatomical characteristics. This has resulted in gaps in our understanding of the genetic control and range of phenotypic variations among different cultivars, and how this diversity relates to overall fitness. Here, we review root anatomical phenotypes in maize and their function in stress adaptation, and briefly discuss phenotyping methods available for root anatomy. We further introduce a simple and accessible phenotyping approach that enables a comprehensive investigation of maize root anatomy. Detailed characterization of root traits and the implementation of robust methods for root anatomical phenotyping could have wide-ranging benefits across various areas of plant science, from fundamental research to enhancing crop breeding efforts.","PeriodicalId":10496,"journal":{"name":"Cold Spring Harbor protocols","volume":" ","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-08-30","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142105085","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Root Anatomy: Preparing, Imaging, and Analyzing Maize Root Cross-Sections. 根部解剖：制备、成像和分析玉米根横截面。

Cold Spring Harbor protocols Pub Date : 2024-08-30 DOI: 10.1101/pdb.prot108585

Jagdeep Singh Sidhu, Hannah M Schneider

{"title":"Root Anatomy: Preparing, Imaging, and Analyzing Maize Root Cross-Sections.","authors":"Jagdeep Singh Sidhu, Hannah M Schneider","doi":"10.1101/pdb.prot108585","DOIUrl":"https://doi.org/10.1101/pdb.prot108585","url":null,"abstract":"Root anatomy plays a critical structural and functional role in the maize root system, and regulates edaphic stress tolerance. The function and genetic basis of several maize root anatomical traits for stress tolerance have been demonstrated. Leveraging root anatomical traits in maize thus holds great potential for developing cultivars with greater nutrient and water efficiency. Key for such approaches is the ability to characterize the root anatomy of plants of interest. Here, we outline a systematic method for preparing, imaging, and analyzing maize root cross-sections. The protocol describes root sectioning (by hand or using a vibratome), preparation of microscope slides and toluidine blue staining, imaging under a light microscope, and both manual and semiautomated methods for anatomical feature extraction from images. The protocol enables the visualization and quantification of various anatomical tissues and traits, and its simplicity, adaptability, and accessibility make it an ideal choice for both small- and large-scale phenotyping studies in maize and other plant species. This standardized protocol provides researchers with a comprehensive methodology to accurately dissect root structures, enabling in-depth analyses that are essential for understanding plant growth, development, and adaptive value for stress tolerance.","PeriodicalId":10496,"journal":{"name":"Cold Spring Harbor protocols","volume":" ","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-08-30","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142105086","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Extraction and Quantification of Total Anthocyanins, Determination of Anthocyanidin Core Structures, and Characterization of Specific Anthocyanins from Maize. 提取和定量总花青素、确定花青素核心结构以及鉴定玉米中的特定花青素。

Cold Spring Harbor protocols Pub Date : 2024-08-26 DOI: 10.1101/pdb.prot108577

Nan Jiang, Lina Gomez, Erich Grotewold

{"title":"Extraction and Quantification of Total Anthocyanins, Determination of Anthocyanidin Core Structures, and Characterization of Specific Anthocyanins from Maize.","authors":"Nan Jiang, Lina Gomez, Erich Grotewold","doi":"10.1101/pdb.prot108577","DOIUrl":"https://doi.org/10.1101/pdb.prot108577","url":null,"abstract":"Anthocyanins are flavonoid pigments that accumulate in fruits and flowers that serve as attractants for pollinators and seed-dispersing organisms. Anthocyanins exhibit diverse chemical structures, characterized both by different anthocyanidin core structures and numerous chemical modifications of the anthocyanidin core. Here, we describe a protocol for the extraction and quantification of total anthocyanins, as well as for the characterization of anthocyanidin core structures and specific anthocyanins, using a spectrophotometer, high-performance liquid chromatography (HPLC), and ultra-high-performance liquid chromatography-two-dimensional mass spectrometry (UHPLC-MS/MS). The method involves anthocyanin extraction using acidic methanol, anthocyanin quantification using a spectrophotometer, determination of anthocyanidin core structure from hydrolyzed anthocyanin extracts using UHPLC-MS/MS, separation of different anthocyanins using HPLC, and characterization of specific anthocyanins using UHPLC-MS/MS. As an example, we describe how we have used this protocol to extract and quantify total anthocyanins from maize leaves, identify cyanidin as the core anthocyanidin structure, and characterize three specific anthocyanins that accumulate in maize leaves, each having a cyanidin core with decorations of a hexose group, and a malonyl or coumaroyl moiety.","PeriodicalId":10496,"journal":{"name":"Cold Spring Harbor protocols","volume":" ","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-08-26","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142072224","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Flavonoids and Derived Anthocyanin Pigments in Plants-Structure, Distribution, Function, and Methods for Quantification and Characterization. 植物中的类黄酮和衍生花青素色素--结构、分布、功能以及定量和表征方法。

Cold Spring Harbor protocols Pub Date : 2024-08-26 DOI: 10.1101/pdb.top108516

Nan Jiang, Erich Grotewold

引用次数: 0

Extraction and Quantification of Phenolic Compounds in Maize. 玉米中酚类化合物的提取与定量

Cold Spring Harbor protocols Pub Date : 2024-08-26 DOI: 10.1101/pdb.prot108576

Lina Gomez, Nan Jiang, Erich Grotewold

{"title":"Extraction and Quantification of Phenolic Compounds in Maize.","authors":"Lina Gomez, Nan Jiang, Erich Grotewold","doi":"10.1101/pdb.prot108576","DOIUrl":"https://doi.org/10.1101/pdb.prot108576","url":null,"abstract":"Plants accumulate hundreds of thousands of specialized metabolites that participate in their interactions with the environment. Among these compounds, phenolics represent a large class, and they play important physiological roles, such as providing a first barrier against pathogens, cues to pollinators, and radiation protection. Maize is one of the most important crops worldwide for food, animal feed, and biofuels, and it has the potential to accumulate different phenolics in vegetative tissues as well as in seeds. Recent studies have identified a large number of phenolic compounds-with a diversity of chemical decorations-in different maize tissues, but these likely represent just a fraction of the metabolic diversity of maize. In this protocol, we describe a specific method for the extraction and quantification of maize phenolic compounds by ultra-high-pressure liquid chromatography-tandem multiple reaction monitoring mass spectrometry (UHPLC-MRM-MS/MS) analysis. We provide detailed instructions for the extraction of phenolics using acidic methanol, and for the quantification of 33 different compounds in maize stems, including flavonoids, phenolic acids, and lignin precursors.","PeriodicalId":10496,"journal":{"name":"Cold Spring Harbor protocols","volume":" ","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-08-26","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142072223","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Tools and Resources at the Maize Genetics and Genomics Database (MaizeGDB). 玉米遗传学和基因组学数据库（MaizeGDB）的工具和资源。

Cold Spring Harbor protocols Pub Date : 2024-08-16 DOI: 10.1101/pdb.over108430

Margaret R Woodhouse, Ethalinda K Cannon, John L Portwood, Jack M Gardiner, Rita K Hayford, Olivia Haley, Carson M Andorf

引用次数: 0

Chicken Immunization followed by RNA Extraction and cDNA Synthesis for Antibody Library Preparation. 鸡免疫后提取 RNA 和 cDNA 合成用于抗体库制备。

Cold Spring Harbor protocols Pub Date : 2024-08-16 DOI: 10.1101/pdb.prot108568

Hyunji Yang, Jisu Chae, Hyori Kim, Jinsung Noh, Junho Chung

{"title":"Chicken Immunization followed by RNA Extraction and cDNA Synthesis for Antibody Library Preparation.","authors":"Hyunji Yang, Jisu Chae, Hyori Kim, Jinsung Noh, Junho Chung","doi":"10.1101/pdb.prot108568","DOIUrl":"https://doi.org/10.1101/pdb.prot108568","url":null,"abstract":"Effective isolation of specific antibodies from immunological repertoires requires the generation of a diverse library against a specific antigen of interest, as well as efficient selection procedures, such as bio-panning and phage ELISA. Key to this is the generation of a good immune response in the host, followed by preparation of high-quality RNA and cDNA from which a library can be constructed by the amplification and cloning of immunoglobulin heavy and light chain genes. The first step in the construction of such an \"immune library\" is a successful course of immunization. Detection of a strong serum antibody titer will theoretically then result in a pool of extracted RNA that is enriched for transcripts of genes encoding the antibody of interest. Chicken antibodies have been widely used for research and diagnostic purposes, largely because of both their cross-reactivity to epitopes shared by humans, mice, primates, and other mammals, and their simple characteristics, with chickens featuring single functional copies of V H /J H and V λ /J λ gene pairs. In chickens, antibodies against an antigen of interest can be detected in the serum as soon as 5-7 d after immunization. Once the antibody titer reaches an appropriate level in the serum, the spleen, bursa of Fabricius, and bone marrow are then harvested, and antibody libraries can be prepared from extracted RNA. Here, we describe a protocol for chicken immunization with an antigen of interest, followed by RNA extraction from the relevant tissues and cDNA synthesis, which users can use for antibody library construction.","PeriodicalId":10496,"journal":{"name":"Cold Spring Harbor protocols","volume":" ","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-08-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141995465","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Preparation of VCSM13 Helper Phage for Display Library Reamplification and Bio-Panning. 制备 VCSM13 辅助噬菌体，用于显示文库重扩增和生物平移。

Cold Spring Harbor protocols Pub Date : 2024-08-16 DOI: 10.1101/pdb.prot108569

Hyunji Yang, Jisu Chae, Hyori Kim, Jinsung Noh, Junho Chung

引用次数: 0

Generation of Chicken Antibody Libraries and Selection of Antigen Binders. 鸡抗体库的生成和抗原结合体的选择。

Cold Spring Harbor protocols Pub Date : 2024-08-16 DOI: 10.1101/pdb.top108210

Hyunji Yang, Jisu Chae, Hyori Kim, Jinsung Noh, Junho Chung

{"title":"Generation of Chicken Antibody Libraries and Selection of Antigen Binders.","authors":"Hyunji Yang, Jisu Chae, Hyori Kim, Jinsung Noh, Junho Chung","doi":"10.1101/pdb.top108210","DOIUrl":"https://doi.org/10.1101/pdb.top108210","url":null,"abstract":"Chicken antibodies have been widely used for research and diagnostic purposes. Chicken antibodies are often cross-reactive to epitopes shared by humans, nonhuman primates, and other mammals, and can be tested in many mouse disease models, which provides an advantage for their preclinical study and evaluation. In addition, the variable region of chicken antibodies has unique structural characteristics, including noncanonical cysteine residues in the heavy chain complementarity-determining region (CDR)3 and a long heavy chain CDR3, which together with a short light chain CDR enable the formation of unconventional antibody paratopes. As chickens have single functional copies of the V H and J H genes, and the somatic gene conversion process usually involves D H genes, all functional VDJ gene fragments can be obtained from the B-cell repertoire using a single PCR primer set, without any primer bias. As for the light chain, chickens only have a V λ light chain, composed of a single V λ and J λ gene pair. Therefore, the chicken light chain repertoire can also be accurately amplified using a single primer set. This unbiased reconstitution of the chicken B-cell repertoire provides a great advantage not only in the construction of phage display libraries but also for the in silico selection of antigen binders from a virtual B-cell receptor repertoire. Here, we introduce the use of chicken antibodies in research, diagnostic, and therapeutic fields. In addition, the chromosomal organization of chicken immunoglobulin genes and its diversification mechanisms for shaping the antibody repertoire are also discussed.","PeriodicalId":10496,"journal":{"name":"Cold Spring Harbor protocols","volume":" ","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-08-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141995467","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Selection of Antigen Binders from a Chicken Single-Chain Variable Fragment Library. 从鸡单链可变片段库中选择抗原结合剂

Cold Spring Harbor protocols Pub Date : 2024-08-16 DOI: 10.1101/pdb.prot108211

Hyunji Yang, Jisu Chae, Hyori Kim, Jinsung Noh, Junho Chung

引用次数: 0