Cold Spring Harbor protocols最新文献_第3页

Experimental Approaches for Assessing Root Mechanical Properties in Maize. 评价玉米根系力学特性的试验方法。

Cold Spring Harbor protocols Pub Date : 2026-08-03 DOI: 10.1101/pdb.top108439

Irene I Ikiriko, Ashley N Hostetler, Erin E Sparks

引用次数: 0

High-Throughput Assay for Measuring Phytate and Available Phosphorus in Ground Maize Seed Samples. 高通量测定玉米种子样品中植酸盐和有效磷的方法。

Cold Spring Harbor protocols Pub Date : 2026-08-03 DOI: 10.1101/pdb.prot108686

Adrienne N Moran Lauter, Anthony Assibi Mahama, M Paul Scott

{"title":"High-Throughput Assay for Measuring Phytate and Available Phosphorus in Ground Maize Seed Samples.","authors":"Adrienne N Moran Lauter, Anthony Assibi Mahama, M Paul Scott","doi":"10.1101/pdb.prot108686","DOIUrl":"10.1101/pdb.prot108686","url":null,"abstract":"Phosphorus is an important nutrient for plants and animals. In maize seeds, phosphorus is stored in the form of phytate, which is the phosphorylated form of the sugar inositol. Monogastric animals lack the enzymes required to break phytate down, so it passes through their digestive systems, to create phosphorus-rich waste. This waste contaminates ground water and leads to water quality problems, such as eutrophication, or excessive concentration of nutrients. Phytate reduces the phosphorus available to animals from their feed, requiring animal feed to be supplemented with phosphate. In addition, phytate chelates nutritionally important metal cations, such as iron and zinc, contributing to globally important nutrient deficiencies in human diets. Development of low-phytate corn is an important breeding objective. To achieve this objective, it is crucial to be able to measure phytate, as well as available phosphorus, in maize seeds. Throughout, precision and cost are important considerations in plant breeding programs. This protocol describes methods for quantifying phytate and available phosphorus in maize seeds, in high-throughput 96 well plate assays, suitable for analysis of large-scale field studies and breeding efforts.","PeriodicalId":10496,"journal":{"name":"Cold Spring Harbor protocols","volume":" ","pages":"pdb.prot108686"},"PeriodicalIF":0.0,"publicationDate":"2026-08-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145854632","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

Three-Point Bend Testing for Quantification of Maize Brace Roots Mechanics. 玉米支撑根力学定量的三点弯曲试验。

Cold Spring Harbor protocols Pub Date : 2026-08-03 DOI: 10.1101/pdb.prot108668

Emilia Pierce, Ashley N Hostetler, Erin E Sparks

引用次数: 0

Grain Quality in Maize. 玉米的籽粒品质。

Cold Spring Harbor protocols Pub Date : 2026-08-03 DOI: 10.1101/pdb.top108476

Anthony Assibi Mahama, Adrienne N Moran Lauter, M Paul Scott

{"title":"Grain Quality in Maize.","authors":"Anthony Assibi Mahama, Adrienne N Moran Lauter, M Paul Scott","doi":"10.1101/pdb.top108476","DOIUrl":"10.1101/pdb.top108476","url":null,"abstract":"Grain quality is defined as the suitability of grain for a particular use. It is usually designated by chemical composition or physical properties of the grain. The ability to measure grain quality is important for identity preservation of specialty grain market classes, for development of new varieties with improved quality through breeding, and for basic scientific studies on the genetic or biochemical control of grain quality traits. This review introduces official methods for measuring maize compositional traits, including protein, starch, oil, amino acid, phytate, and phosphorus content. Additionally, we discuss two nonofficial methods: measuring phytate and available phosphorus levels, and assessing amino acid balance. Phytate and available phosphorous impact the mineral nutrition of grain, while amino acid balance reflects the value of grain as a protein source and the bioavailability of protein. We also describe the use of near-infrared spectroscopy (NIRS) to assess levels of various compounds in maize. NIRS relies on the fact that compounds with differing molecular properties uniquely interact with the near-infrared region (750-2500 nm) of the electromagnetic radiation spectrum, and thus, generate spectral information that can be used to develop calibration models/equations for predicting the concentration of the compounds in grain samples. We discuss how sensitivity, accuracy, precision, throughput, and cost influence the choice of assay used to assess grain quality. Furthermore, we discuss how appropriate experimental design and data analysis can improve analytical outcomes when assessing grain quality.","PeriodicalId":10496,"journal":{"name":"Cold Spring Harbor protocols","volume":" ","pages":"pdb.top108476"},"PeriodicalIF":0.0,"publicationDate":"2026-08-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145854623","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

In-Field Evaluation of Root Lodging Resistance of Whole Maize Plants. 玉米全株抗倒伏性的田间评价。

Cold Spring Harbor protocols Pub Date : 2026-08-03 DOI: 10.1101/pdb.prot108669

Jinzhong Yang, Juan Liu, Jinsheng Yang, Xiaohu Wang, Yinchang Li

{"title":"In-Field Evaluation of Root Lodging Resistance of Whole Maize Plants.","authors":"Jinzhong Yang, Juan Liu, Jinsheng Yang, Xiaohu Wang, Yinchang Li","doi":"10.1101/pdb.prot108669","DOIUrl":"10.1101/pdb.prot108669","url":null,"abstract":"Root lodging due to wind is common in maize production worldwide, and can reduce photosynthetic capacity as well as nutrient uptake, resulting in significant yield loss and seed quality reduction. Lodging also causes harvesting problems, and ultimately increases production costs. Evaluating maize resistance to lodging is thus important for both breeders and researchers, to optimize agricultural practices, enhance breeding strategies, and ultimately develop new maize varieties with improved resilience. Here, we describe a novel procedure to accurately and quantitatively assess the resistance of maize plants to root lodging in the field. In this approach, users measure mechanical properties of maize root systems and estimate the magnitude of the wind force acting on the maize plants to ultimately derive an antilodging index, a measure that thus considers the balance between internal and external forces acting on the plants in the field. The procedure, which focuses on the plant as a whole and not only on the root system, has been successfully used to evaluate lodging resistance throughout the entire growth period, from the V8 growth stage to plant maturity, in different maize genotypes. We also compare the procedure to others in the literature, and discuss its applicability for assessing crop root lodging resistance in breeding and cultivation programs.","PeriodicalId":10496,"journal":{"name":"Cold Spring Harbor protocols","volume":" ","pages":"pdb.prot108669"},"PeriodicalIF":0.0,"publicationDate":"2026-08-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145298987","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

High-Throughput Microbial Assay for Amino Acid Measurement in Ground Maize Seed Samples Utilizing Auxotrophic E. coli. 利用营养不良大肠杆菌测定碾碎玉米种子样品中氨基酸的高通量微生物试验。

Cold Spring Harbor protocols Pub Date : 2026-08-03 DOI: 10.1101/pdb.prot108687

Adrienne N Moran Lauter, Anthony Assibi Mahama, M Paul Scott

{"title":"High-Throughput Microbial Assay for Amino Acid Measurement in Ground Maize Seed Samples Utilizing Auxotrophic E. coli.","authors":"Adrienne N Moran Lauter, Anthony Assibi Mahama, M Paul Scott","doi":"10.1101/pdb.prot108687","DOIUrl":"10.1101/pdb.prot108687","url":null,"abstract":"Amino acids are important nutrients in maize grain used for food and feed. Because all 20 amino acids are required for growth and development, a deficiency in a single essential amino acid limits the utilization of dietary protein. In monogastric animals, 10 amino acids must be supplied by the diet and therefore are considered essential. The remaining amino acids can be made from the 10 essential amino acids. Lysine, tryptophan, and methionine are frequently limiting essential amino acids in grain-based diets. Therefore, increasing levels of limiting essential amino acids in grain is an important objective in crop improvement. Standard chromatographic methods for assessing levels of amino acids in grain are extremely accurate, but very expensive. Here, we present a protocol for high-throughput analysis of amino acids in grains, using microbial assays, conducted in 96 well plates, that can be carried out for a fraction of the cost of the standard chromatographic methods. We use Escherichia coli strains that have mutations in the biosynthetic pathway of the amino acid of interest. These strains are auxotrophic, so their growth is proportional to the amount of a specific amino acid in the media. The level of the amino acid of interest in a corn extract is determined by adding the corn extract to the microbial growth medium and measuring the growth of the culture as turbidity in a 96 well plate reader. This protocol is designed for analysis of methionine, but can be adapted for the analysis of any amino acid, by substitution of an appropriate auxotrophic strain of E. coli.","PeriodicalId":10496,"journal":{"name":"Cold Spring Harbor protocols","volume":" ","pages":"pdb.prot108687"},"PeriodicalIF":0.0,"publicationDate":"2026-08-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145854635","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

Advances in Phage Display-A Perspective. 噬菌体显示研究进展

Cold Spring Harbor protocols Pub Date : 2026-07-01 DOI: 10.1101/pdb.over107753

George P Smith

{"title":"Advances in Phage Display-A Perspective.","authors":"George P Smith","doi":"10.1101/pdb.over107753","DOIUrl":"10.1101/pdb.over107753","url":null,"abstract":"Phage display technology is enabled by genetic fusion of a foreign protein domain to a phage coat protein, without interfering with the phage's ability to replicate by infecting bacterial host cells. The displayed domain is exposed on the phage particle (virion) surface, where it can interact with molecules or other substances in the surrounding medium; in this regard, it acts like a normal protein. However, it possesses a superpower that is unavailable to ordinary proteins: It is easily replicated in great abundance because it is attached to a replicating virion whose genome includes its coding sequence. The main way this technology is exploited is construction of huge phage display \"libraries,\" comprising billions of phage clones, each displaying a different protein domain, and each represented by thousands, millions, or billions of genetically identical virions-all mixed together in a single vessel. Surface display allows exceedingly rare virions whose displayed protein domains happen to bind a user-defined molecule or other substance-generically called the \"selector\"-to be isolated from such libraries by an affinity selection process. The yield of selector-binding virions is much too low to be of practical use, but their number is readily increased by many orders of magnitude by propagating the virions in host bacteria in culture. This overview is a critical review of recent developments of this technology. It does not review the entire arena of contemporary phage display; there is special emphasis on phage display's most prominent application, phage antibodies, in which the displayed domain is an antibody domain, and the selector is an antigen of interest.","PeriodicalId":10496,"journal":{"name":"Cold Spring Harbor protocols","volume":" ","pages":"pdb.over107753"},"PeriodicalIF":0.0,"publicationDate":"2026-07-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145343774","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

Cloning and Selection from Antigen Fragment Libraries for Epitope Identification. 抗原片段文库的克隆与选择与表位鉴定。

Cold Spring Harbor protocols Pub Date : 2026-07-01 DOI: 10.1101/pdb.prot108660

Gregg J Silverman

{"title":"Cloning and Selection from Antigen Fragment Libraries for Epitope Identification.","authors":"Gregg J Silverman","doi":"10.1101/pdb.prot108660","DOIUrl":"10.1101/pdb.prot108660","url":null,"abstract":"To understand what drives an immune response, it is important to characterize, at a molecular level, the site(s) on an immunogenic antigen that is directly contacted by a soluble antibody or B-cell antigen receptor (BCR) on the surface of a B lymphocyte. Moreover, antibody binding interactions with a microbial protein can interfere with the functional activity of a toxin (i.e., neutralization) and/or can aid in the clearance of the microbial protein from the body, further underscoring the importance of such characterization. Phage display technology is a potent tool that can be used to study any type of protein-protein interaction. In recent years, we have refined methods for the identification of the minimal binding contact sites of an antibody with an antigen. Here, we describe a workflow for optimizing antibody-mediated selection and for the identification and characterization of antigen-specific epitopes. This workflow includes (1) the generation of large libraries of random fragments of a gene of interest cloned into the validated pComb-Opti8 phagemid expression cloning vector system; (2) electroporation of these libraries into electrocompetent bacterial cells and subsequent recovery of viral particles, each of which displays the cloned gene fragment product as a fusion protein with the filamentous phage major coat protein VIII (pVIII); (3) recovery of individual phagemid clones that express the smallest functional epitopes recognized by an experimental antibody; (4) an efficient means of using high-throughput DNA sequencing to interrogate sequentially selected libraries to rapidly identify the gene subregions encoding epitopes of interest; and (5) means for the further characterization of potential antibody-epitope binding interactions.","PeriodicalId":10496,"journal":{"name":"Cold Spring Harbor protocols","volume":" ","pages":"pdb.prot108660"},"PeriodicalIF":0.0,"publicationDate":"2026-07-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145470605","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

CRISPR-Cas9 Toolkit for Maize: Vector Design, Construction, and Analysis of Edited Plants. 玉米CRISPR-Cas9工具箱：编辑植物的载体设计、构建和分析

Cold Spring Harbor protocols Pub Date : 2026-07-01 DOI: 10.1101/pdb.prot108659

Si Nian Char, Hua Liu, James A Birchler, Kan Wang, Bing Yang

{"title":"CRISPR-Cas9 Toolkit for Maize: Vector Design, Construction, and Analysis of Edited Plants.","authors":"Si Nian Char, Hua Liu, James A Birchler, Kan Wang, Bing Yang","doi":"10.1101/pdb.prot108659","DOIUrl":"10.1101/pdb.prot108659","url":null,"abstract":"Genetic toolsets are essential for gene discovery, elucidating biological pathways, and accelerating molecular breeding of superior crops in plant biology and agriculture. Among these, the CRISPR-Cas9 (clustered regularly interspaced short palindromic repeats-CRISPR-associated protein 9) system has emerged as a powerful and indispensable tool for precise genome editing in maize (Zea mays L.). This protocol presents a comprehensive, maize-specific approach to constructing CRISPR vectors and analyzing transgenic plants carrying targeted gene mutations. It is organized into two major sections. The first section provides a step-by-step guide for designing guide RNAs and oligonucleotides (oligos) to construct CRISPR vectors containing one, two, four, or multiplexed (up to eight) single-guide RNAs (sgRNAs). It also describes the modular assembly of these sgRNAs with the Cas9 expression cassette using the Gateway cloning strategy to streamline vector construction. The second section focuses on genotyping CRISPR-edited plants by detecting and characterizing target mutations. Four complementary methods are outlined: (1) the T7 endonuclease I (T7EI) assay, (2) restriction enzyme digestion, (3) Sanger sequencing of PCR amplicons, and (4) high-throughput sequencing. Methods 1 and 2 offer rapid and cost-effective screening for small insertions or deletions (indels), while methods 3 and 4 provide high-resolution and scalable mutation analysis. Together, this workflow offers researchers an efficient, flexible, and reliable system for genome editing and mutation validation in maize, supporting both functional genomics studies and trait improvement applications.","PeriodicalId":10496,"journal":{"name":"Cold Spring Harbor protocols","volume":" ","pages":"pdb.prot108659"},"PeriodicalIF":0.0,"publicationDate":"2026-07-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145243798","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

Insights from the Study of B-Cell Epitopes of a Microbial Pathogen by Phage Display. 利用噬菌体展示技术研究病原菌b细胞表位的新发现。

Cold Spring Harbor protocols Pub Date : 2026-07-01 DOI: 10.1101/pdb.top107777

Gregg J Silverman

{"title":"Insights from the Study of B-Cell Epitopes of a Microbial Pathogen by Phage Display.","authors":"Gregg J Silverman","doi":"10.1101/pdb.top107777","DOIUrl":"10.1101/pdb.top107777","url":null,"abstract":"The human immune system evolved to defend against the panoply of microbial threats. By harnessing such ability, vaccines have cumulatively saved hundreds of millions of lives. Despite such tremendous success, there have also been remarkable failures, such as the lack of a clinically proven vaccine against Staphylococcus aureus (SA), which continues to pose an urgent public health threat. In practice, it has proven challenging to identify the molecular basis for relevant epitopes for this pathogen. Here, we summarize our experience implementing an integrated approach using phage display technology for the identification of B-cell epitopes of microbial virulence factors, which we developed with a focus on SA. This approach was used to define minimal B-cell epitopes of the staphylococcal leucocidin family of pore-forming toxins (PFTs) that have been implicated in staphylococcal clinical infection. Our methodology provides proof of principle for an approach well suited for the rapid and efficient generation of modular protein-based vaccines for protection from clinical infection, which can be used to target pathogens for which no vaccine is currently available.","PeriodicalId":10496,"journal":{"name":"Cold Spring Harbor protocols","volume":" ","pages":"pdb.top107777"},"PeriodicalIF":0.0,"publicationDate":"2026-07-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145470632","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