Current protocols in plant biology最新文献

Maize B73 is a reference genome and has long been a major resource for genetics and molecular biology research. We have developed an efficient B73 transformation protocol by enabling somatic embryogenesis through differential co-expression of maize morphogenic regulators BBM and WUS2. We describe a successful protocol that utilizes Agrobacterium tumefaciens strain AGL1 harboring binary vector PHP78891 that comprises a BBM and WUS2 expression cassette as well as a green fluorescent protein (GFP) reporter cassette. The PHP78891 vector also contains, within the T-DNA region, a CRE/lox recombination system flanking the CRE/BBM/WUS2 co-expression cassette driven by the desiccation inducible RAB17 promoter that allows removal of the BBM/WUS2 cassette. Introduction and co-expression of BBM and WUS2 induced direct somatic embryogenesis (SE) in non-regenerable maize B73 from immature embryo explants. Removal of the CRE/BBM/WUS2 cassette is essential to allow regeneration to fertile plants. The GFP expression cassette outside the lox excision sites is retained in the transgenic plant genome, allowing subsequent phenotypic analysis of calli and regenerated transgenic events. This transformation system enables a selectable marker-free transformation process by taking advantage of BBM/WUS2-induced SE as a developmental selection system. © 2018 by John Wiley & Sons, Inc.

Most reliable transformation protocols for cereal crops, including sorghum (Sorghum bicolor L. Moench), rely on the use of immature embryo explants to generate embryogenic callus cells that are then transformed using Agrobacterium- or particle-bombardment-mediated DNA delivery. Subsequent to DNA transfer, most protocols rely on selectable markers for the recovery of stably transformed callus that is then regenerated to produce T₀ plants. However, these protocols require specific genotypes that are innately capable of efficient embryogenic callus initiation. Here, we describe a system that makes use of the differential expression of the morphogenic regulators Baby Boom (Bbm) and Wuschel2 (Wus2) to achieve transformation in varieties of sorghum typically recalcitrant to standard transformation methods. © 2018 by John Wiley & Sons, Inc.

Genetic transformation via Agrobacterium-mediated methodology has been used in many sorghum studies. However, the transformation efficiency still varies significantly due to high dependence on sorghum genotypes and technical expertise. In this article, we describe a sorghum transformation procedure in sufficient detail using a public genotype, P898012. This system utilizes a standard binary transgenic vector carrying the bar gene as a selectable marker and immature embryos as starting explants. Glufosinate is employed as the selective agent during callus and shoot induction. This procedure is relatively rapid, efficient, highly reproducible, and should be applicable for many other sorghum genotypes. © 2018 by John Wiley & Sons, Inc.

Interactions occurring between DNA and proteins across the nuclear genome regulate numerous processes, including meiosis. Meiosis ensures genetic variation and balanced segregation of homologous chromosomes. It involves complex DNA-protein interactions across the entire genome to regulate a broad range of processes, including formation and repair of double-strand DNA breaks (DSBs), chromosome compaction, homolog pairing, synapsis, and homologous recombination. The latter meiotic event, meiotic recombination, often occurs at discrete locations in a genome, within a tight time window. The identification of genomic binding sites of meiotic proteins is a major step toward understanding the molecular mechanisms underlying meiotic recombination and provides important information for plant breeding. Collecting meiotic cells from plants is challenging, tedious, and time consuming, since the meiocyte-producing organs, the anthers, are generally small and limited to certain developmental stages of plants. Here we provide a protocol to isolate meiotic-stage-specific anthers and perform ChIP on this material. We have developed a ChIP protocol specifically suited to (1) small amounts of input material and (2) proteins that bind transiently to chromatin and at very low frequency. © 2018 by John Wiley & Sons, Inc.

The human body contains approximately 3.2% nitrogen (N), mainly present as protein and amino acids. Although N exists at a high concentration (78%) in the air, it is not readily available to animals and most plants. Plants are however able to take up both nitrate (NO₃⁻) and ammonium (NH₄⁺) ions from the soil and convert them to amino acids and proteins, which are excellent sources for all animals. Most N is available as the stable isotope ¹⁴N, but a second form, ¹⁵N, is present in very low concentrations. ¹⁵N can be detected in extracts of plants by gas chromatography followed by mass spectrometry (GC/MS). In this protocol, the methods are described for tracing the pathway by which plants are able to take up ¹⁵N-labeled nitrate and ammonium and convert them into amino acids and proteins. A protocol for extracting and quantifying amino acids and ¹⁵N enrichment in maize (Zea mays L.) leaves labeled with ¹⁵NH₄⁺ is described. Following amino acid extraction, purification, and separation by GC/MS, a calculation of the ¹⁵N enrichment of each amino acid is carried out on a relative basis to identify any differences in the dynamics of amino acid accumulation. This will allow a study of the impact of genetic modifications or mutations on key reactions involved in primary nitrogen and carbon metabolism. © 2018 by John Wiley & Sons, Inc.

Maize is one the most widely cultivated crops worldwide and an important model system for the study of genetics and cytogenetics. Although the availability of a genome sequence has enabled new quantitative genomic studies, developing methods to isolate specific types of cells will enable useful approaches for transcriptomic analysis in the crop plant. Fluorescence-activated cell sorting (FACS) is a powerful technique for cell isolation and the study of transcriptional profiles from specific cell populations. The use of FACS on plant cells requires the generation of protoplasts by tissue digestion and cell wall removal. Although some protocols are available, they mainly focus on dicot species and obtaining sufficient protoplasts from inner tissue layers has been challenging in both monocots and dicots. Here, we report a new protocol that dramatically increases protoplast yield from maize for subsequent cell isolation by FACS. This protocol is efficient in generating protoplasts from root and shoot inner layers and can also be applied successfully to Arabidopsis thaliana. © 2018 by John Wiley & Sons, Inc.

Physical interactions between proteins and other molecules can be evaluated at a proteome scale using protein arrays, a type of high-throughput pulldown assay. We developed a modified in situ protein array known as the nucleic acid programmable protein assay (NAPPA) that allows the screening of thousands of open reading frames (ORFs) at a lower cost, with less labor, and in less time than conventional protein arrays. The HaloTag-NAPPA protein array can efficiently capture proteins expressed in situ on a glass slide using the Halo high-affinity capture tag. Here, we describe the fabrication of the array using publicly available resources and detection of protein-protein interactions (PPIs) that can be used to generate a protein interactome map. The Basic Protocol includes procedures for preparing the plasmid DNA spotted on glass slides, in situ protein expression, and PPI detection. The supporting protocols outline the construction of vectors and preparation of ORF clones. © 2018 by John Wiley & Sons, Inc.

In this article, we describe a Y2H interaction mapping protocol for systematically screening medium-to-large collections of open reading frames (ORFs) against each other. The protocol is well suited for analysis of focused networks, for modules of interest, assembling genome-scale interactome maps, and investigating host-microbe interactions. The four-step high-throughput protocol has a demonstrated low false-discovery rate that is essential for producing meaningful network maps. Following the assembly of yeast expression clones from an existing ORFeome collection, we describe in detail the four-step procedure that begins with the primary screen using minipools, followed by secondary verification of primary positives, identification of candidate interaction pairs by sequencing, and a final verification step using fresh inoculants. In addition to this core protocol, aspects of network mapping and quality control will be discussed briefly. © 2018 by John Wiley & Sons, Inc.

A quarter of a century has passed since Lotus japonicus was proposed as a model legume because of its suitability for molecular genetic studies. Since then, a comprehensive set of genetic resources and tools has been developed, including recombinant inbred lines, a collection of wild accessions, published mutant lines, a large collection of mutant lines tagged with LORE1 insertions, cDNA clones with expressed sequence tag (EST) information, genomic clones with end-sequence information, and a reference genome sequence. Resource centers in Japan and Denmark ensure easy access to data and materials, and the resources have greatly facilitated L. japonicus research, thereby contributing to the molecular understanding of characteristic legume features such as endosymbiosis. Here, we provide detailed instructions for L. japonicus cultivation and describe how to order materials and access data using the resource center websites. The comprehensive overview presented here will make L. japonicus more easily accessible as a model system, especially for research groups new to L. japonicus research. © 2018 by John Wiley & Sons, Inc.

In vivo isotopic labeling empowers proteomic and metabolomic analyses to resolve relationships between the molecular composition, environment, and phenotype of an organism. Carbon-13 is particularly useful for plant labeling as it can be introduced via ¹³CO₂ gas and readily assimilated into plant metabolic systems through natural carbon fixation. While short-term labeling experiments can be performed within a simple sealed enclosure, long-term growth in an isolated environment raises many challenges beyond nutrient availability and buildup of metabolic waste. Viable growth conditions must be maintained by means that do not compromise the integrity of the carbon-13 enrichment. To address these issues, an automated growth chamber equipped with countermeasures to neutralize stresses and ensure high isotopic enrichment throughout the life cycle of the plant has been developed. The following describes this growth chamber and its use in an example 130-day growth of ten soybean plants to full maturity, achieving 100% carbon-13 enrichment of new seed tissue. © 2018 by John Wiley & Sons, Inc.