Recombinant Oryza sativa subsp. japonica Barley B recombinant-like protein C (Os10g0115500, LOC_Os10g02620)

What is Os10g0115500 and what functional domains does it contain?

Os10g0115500 (LOC_Os10g02620) encodes the Barley B recombinant-like protein C (BBR), a transcription factor in Oryza sativa subspecies japonica. The protein contains a DNA binding domain known as the BBR domain spanning positions 54-343 . It belongs to the GAGA_bind (PF06217) Pfam family with InterPro identification number IPR010409 .

The protein sequence of the BBR domain is characterized by several histidine-rich regions and DNA-binding motifs that enable it to recognize specific DNA sequences, particularly GA-rich octadinucleotide repeats . This binding activity allows BBR to participate in transcriptional regulation of homeobox genes and influence leaf development in rice .

How is Os10g0115500 related to homologous proteins in other species?

Os10g0115500 has high sequence conservation across multiple rice species and other cereals, suggesting important evolutionary conservation. The similarity scores between this protein and its homologs reveal its evolutionary relationships:

This high level of conservation across grass species suggests that BBR proteins evolved in the common ancestor of Poaceae and have maintained their functional importance throughout evolution . The conservation pattern indicates strong selective pressure, implying that BBR controls fundamental aspects of grass development. This conservation makes BBR an excellent candidate for studying the evolution of transcriptional regulation in plants.

How should I design experiments to study BBR's DNA-binding specificity?

To characterize BBR's DNA-binding specificity, implement a multi-tiered experimental approach:

In vitro binding assays:

• Express recombinant BBR protein using E. coli expression systems (BL21(DE3) strain recommended)

• Purify using His-tag affinity chromatography (Ni-NTA or Co-NTA resins)

• Perform Electrophoretic Mobility Shift Assays (EMSA) with labeled DNA fragments containing GA octadinucleotide repeats

• Use DNase I footprinting to identify protected nucleotides

Chromatin Immunoprecipitation (ChIP):

• Generate antibodies against BBR or use epitope-tagged BBR expressed in rice

• Cross-link protein-DNA complexes in vivo

• Fragment chromatin and immunoprecipitate BBR-bound fragments

• Sequence precipitated DNA (ChIP-seq) to identify genome-wide binding sites

Systematic Evolution of Ligands by Exponential Enrichment (SELEX):

• Incubate purified BBR with random DNA oligonucleotides

• Isolate bound sequences and amplify through PCR

• Repeat multiple times to enrich for high-affinity binding sequences

• Sequence the enriched pool to derive consensus binding motifs

Remember that experimental conditions may need optimization. For instance, buffer composition, salt concentration, and pH can significantly affect binding specificity. Include appropriate positive and negative controls, and validate findings through multiple independent techniques.

What are the key considerations for designing knockout or knockdown experiments for Os10g0115500?

When designing genetic modification experiments to study BBR function, consider these critical factors:

Choice of genetic modification approach:

• CRISPR/Cas9 knockout: Design guide RNAs targeting early exonic regions of Os10g0115500

• RNAi knockdown: Design hairpin constructs targeting unique regions of BBR mRNA

• TALEN approach: Design nucleases targeting specific sequences in the BBR gene

Transformation strategy:

• Use Agrobacterium-mediated transformation for stable integration

• Include appropriate selection markers (hygromycin, kanamycin) in transformation vectors

• Design PCR primers for genotyping to identify successfully edited plants

Experimental controls:

• Include wild-type plants grown under identical conditions

• Use plants transformed with non-targeting constructs as additional controls

• Consider creating multiple independent transgenic lines to control for position effects

Phenotypic analysis approach:

• Focus primarily on leaf morphology and development (based on BBR's known function)

• Perform comprehensive phenotyping across different developmental stages

• Document changes in plant architecture, flowering time, and reproductive success

• Use quantitative measurements (leaf dimensions, angles, curvature) rather than qualitative assessments

Molecular confirmation:

• Verify knockout/knockdown through RT-qPCR to confirm reduced transcript levels

• Perform Western blotting to confirm protein reduction/absence

• Include complementation studies by reintroducing wild-type BBR to confirm phenotype rescue

This comprehensive experimental design will help establish causality between BBR function and observed phenotypes while minimizing confounding variables and experimental artifacts.

How can I analyze differential gene expression between wild-type and BBR-deficient rice plants?

A robust approach to analyze transcriptional changes in BBR-deficient plants:

Sample collection and preparation:

• Collect tissues at relevant developmental stages, focusing on actively growing leaf tissue

• Use at least 3-4 biological replicates per genotype

• Extract high-quality RNA (RIN > 8) using standard methods

• Prepare RNA-seq libraries with polyA selection or rRNA depletion

Sequencing considerations:

• Aim for 20-30 million paired-end reads per sample

• Use appropriate sequencing depth based on rice transcriptome complexity

• Include spike-in controls for normalization

Data analysis pipeline:

• Quality control: Use FastQC to assess read quality

• Read processing: Trim adapters and low-quality bases using Trimmomatic

• Alignment: Map to the rice reference genome using HISAT2 or STAR

• Quantification: Count reads per gene using featureCounts or HTSeq

• Differential expression: Use DESeq2 or edgeR with FDR < 0.05

Statistical approach:

• Implement a negative binomial model for count data

• Control for batch effects using ComBat or RUVSeq if necessary

• Apply multiple testing correction (Benjamini-Hochberg procedure)

• Consider both statistical significance and fold change magnitude

Functional interpretation:

• Perform Gene Ontology enrichment analysis on differentially expressed genes

• Identify enriched biological pathways using KEGG or Plant Reactome

• Compare with known BBR targets or genes involved in leaf development

• Visualize results using volcano plots, heatmaps, and pathway diagrams

Validation:

• Select 8-10 differentially expressed genes for validation via RT-qPCR

• Include genes with varying expression levels and fold changes

• Use stable reference genes for normalization (e.g., UBQ, Actin)

This approach will provide a comprehensive view of transcriptional changes resulting from BBR deficiency and help identify direct and indirect targets of this transcription factor.

How does BBR influence leaf morphology and development?

BBR influences leaf morphology through its role as a transcription factor regulating homeobox genes involved in leaf development . To fully characterize this function, implement the following integrated approach:

Morphological analysis:

• Compare leaf dimensions (length, width, area) between wild-type and BBR-deficient plants

• Analyze leaf curvature, venation patterns, and cell arrangement using microscopy

• Examine leaf developmental stages to identify when BBR function is most critical

• Quantify changes in leaf shape using computational morphometrics

Molecular characterization:

• Identify BBR-regulated homeobox genes through ChIP-seq and RNA-seq

• Validate direct regulation through reporter assays and EMSA

• Perform epistasis experiments with known leaf development genes

• Analyze the expression patterns of BBR during leaf development stages

Cellular analysis:

• Compare cell size, number, and arrangement in BBR-deficient versus wild-type leaves

• Analyze patterns of cell division and expansion during leaf development

• Investigate potential roles in adaxial-abaxial polarity establishment

• Determine if BBR affects symmetry or asymmetry in leaf development

Hormonal interactions:

• Examine BBR's relationship with plant hormones involved in leaf development (auxin, cytokinin)

• Test BBR-deficient plants' responses to exogenous hormone treatments

• Analyze expression of hormone biosynthesis and signaling genes in BBR mutants

This multi-faceted approach will provide mechanistic insights into how BBR regulates leaf development at molecular, cellular, and organismal levels.

What is the best approach for constructing a regulatory network centered on BBR?

To construct a comprehensive BBR-centered regulatory network, integrate multiple data types using this systematic approach:

Data generation:

• Perform ChIP-seq to identify direct BBR binding sites genome-wide

• Conduct RNA-seq comparing wild-type and BBR-deficient plants

• Generate time-course data to capture primary vs. secondary effects

• Include ATAC-seq to identify changes in chromatin accessibility

Integrative analysis:

• Assign ChIP-seq peaks to potential target genes (typically within 2kb of TSS)

• Intersect differentially expressed genes with genes having BBR binding sites

• Categorize direct targets as activated (downregulated in knockout) or repressed (upregulated)

• Use gene regulatory network inference algorithms (GENIE3, ARACNE) to identify co-regulated genes

Network validation:

• Validate key regulatory connections through reporter assays

• Perform ChIP-qPCR to confirm binding at selected loci

• Use transient expression systems to verify functional relationships

• Implement CRISPR interference at selected binding sites to test functionality

Network visualization and analysis:

• Use Cytoscape for network visualization with BBR as central hub

• Identify network motifs (feed-forward loops, feedback loops)

• Apply modularity analysis to identify functional modules

• Compare with known developmental regulatory networks in plants

Integration with existing knowledge:

• Incorporate known interactions from literature

• Add protein-protein interaction data if available

• Compare with regulatory networks of related transcription factors

• Annotate with Gene Ontology terms to identify biological processes

This approach will yield a comprehensive view of BBR's regulatory role and place it within the broader context of transcriptional networks governing rice development.

What expression systems are optimal for producing recombinant BBR protein?

Multiple expression systems can be used for BBR protein production, each with advantages for specific applications:

E. coli expression system:

• Recommended strains: BL21(DE3), Rosetta-GAMI

• Advantages: High yield, simple protocols, cost-effective

• Optimal for: Initial biochemical characterization, DNA binding studies

• Expression optimization: Use 0.1-0.5 mM IPTG, induce at 18-25°C for improved solubility

• Typical yield: 5-10 mg/L culture

Yeast expression system:

• Recommended strains: SMD1168, GS115, X-33

• Advantages: Better protein folding, moderate post-translational modifications

• Optimal for: When E. coli expression yields insoluble protein

• Expression strategy: Strong inducible promoters (AOX1, GAL1)

• Typical yield: 1-5 mg/L culture

Insect cell expression system:

• Recommended cell lines: Sf9, Sf21, High Five

• Advantages: Complex eukaryotic processing, good for proteins requiring chaperones

• Optimal for: Structural studies requiring properly folded protein

• Expression vector: Baculovirus with polyhedrin or p10 promoter

• Typical yield: 1-10 mg/L culture

Mammalian cell expression system:

• Recommended cell lines: HEK293, CHO

• Advantages: Most sophisticated post-translational modifications

• Optimal for: Functional studies requiring authentic modifications

• Expression strategy: Transient or stable expression with CMV promoter

• Typical yield: 0.1-1 mg/L culture

Fusion tag recommendations:

• For solubility: MBP or GST tags

• For purification: His-tag (minimal interference with structure)

• For detection: FLAG tag or GFP

• Tag position considerations: N-terminal tags often preserve C-terminal DNA binding activity

For most applications, start with E. coli expression using a His-tag construct, optimizing temperature and inducer concentration to maximize soluble protein production .

What purification strategies yield the highest purity BBR protein?

To obtain high-purity BBR protein suitable for functional and structural studies, implement this multi-step purification strategy:

Initial capture:

• Immobilized Metal Affinity Chromatography (IMAC) for His-tagged BBR

• Use Ni-NTA or Co-NTA resin with imidazole gradient elution

• Typical conditions: 50 mM Na₂HPO₄ pH 7.4, 0.5 M NaCl, elute with 50-500 mM imidazole

• Consider adding 5-10% glycerol and 1-5 mM DTT to maintain stability

Intermediate purification:

• Ion exchange chromatography to remove DNA and other contaminants

• For BBR (predicted pI ~8-9), use cation exchange at pH 7.0

• Elute with salt gradient (50-1000 mM NaCl)

• This step is crucial for removing DNA bound to this transcription factor

Tag removal:

• Include protease cleavage site between tag and BBR (TEV or PreScission)

• Perform cleavage at 4°C overnight with optimized protease:protein ratio

• Remove cleaved tag by reverse IMAC

• Monitor cleavage efficiency by SDS-PAGE

Polishing step:

• Size exclusion chromatography for homogeneous preparation

• Recommended columns: Superdex 75 or 200 (depending on oligomeric state)

• Running buffer: 20 mM Tris pH 7.5, 150 mM NaCl, 1 mM DTT, 5% glycerol

• Analyze elution profile for monodispersity

Quality control:

• Assess purity by SDS-PAGE (aim for >95%)

• Verify identity by mass spectrometry

• Evaluate homogeneity by dynamic light scattering

• Test DNA-binding activity by EMSA

This purification workflow balances yield and purity considerations while preserving the functional integrity of the BBR protein.

How can I design primers for amplifying and cloning Os10g0115500?

Designing effective primers for cloning and expressing Os10g0115500 requires careful consideration of multiple factors:

Complete CDS primer design:

BBR domain-specific primers (positions 54-343):

PCR optimization recommendations:

• Use high-fidelity DNA polymerase (Q5, Phusion, or PfuUltra)

• Initial denaturation: 98°C for 30 seconds

• 25-30 cycles of:

  • Denaturation: 98°C for 10 seconds

  • Annealing: 58-62°C for 30 seconds (run gradient PCR to optimize)

  • Extension: 72°C for 1 minute (30 seconds/kb)

• Final extension: 72°C for 5 minutes

• Add 3-5% DMSO if GC-rich regions cause amplification problems

Cloning strategy considerations:

• Select a vector compatible with your expression system and fusion tag requirements

• Perform restriction digestion of PCR product and vector using the same enzymes

• Gel-purify digested fragments before ligation

• Transform into high-efficiency competent cells

• Screen colonies by colony PCR and confirm by sequencing

This comprehensive primer design strategy will facilitate successful cloning of Os10g0115500 for various experimental applications.

How does BBR interact with the chromatin landscape to regulate gene expression?

Understanding BBR's interaction with chromatin requires integrated genomic approaches:

Chromatin accessibility analysis:

• Perform ATAC-seq or DNase-seq in wild-type and BBR-deficient plants

• Compare accessible chromatin regions to identify BBR-dependent changes

• Overlay BBR binding sites with accessibility data to determine if BBR promotes opening or closing of chromatin

• Analyze DNA methylation patterns at BBR binding sites

Histone modification analysis:

• Conduct ChIP-seq for key histone marks (H3K4me3, H3K27ac, H3K27me3) in wild-type and BBR mutants

• Determine if BBR influences active (H3K4me3, H3K27ac) or repressive (H3K27me3) modifications

• Analyze temporal dynamics of histone modifications following BBR binding

• Investigate potential interactions between BBR and histone-modifying enzymes

Chromatin remodeler interactions:

• Test for physical interactions between BBR and chromatin remodeling complexes via co-IP

• Compare BBR binding sites with known locations of chromatin remodelers

• Determine if BBR recruits specific remodeling complexes to target sites

• Analyze nucleosome positioning around BBR binding sites

3D chromatin organization:

• Implement Hi-C or Micro-C to determine if BBR affects higher-order chromatin structure

• Identify potential enhancer-promoter interactions mediated by BBR

• Compare topologically associated domains (TADs) between wild-type and BBR mutants

• Use Capture-C to focus on specific BBR-regulated loci

This multi-layered approach will reveal how BBR functions within the broader chromatin regulatory landscape to control gene expression in rice.

What evolutionary insights can be gained from comparative analysis of BBR across species?

A comprehensive evolutionary analysis of BBR can reveal fundamental insights about its function and conservation:

Sequence evolution analysis:

• Perform phylogenetic analysis of BBR proteins across plants (focus on Poaceae)

• Calculate selection pressure (dN/dS ratios) across different protein domains

• Identify regions under purifying selection (functionally constrained) versus diversifying selection

• Reconstruct ancestral BBR sequences to infer the evolutionary history of this transcription factor

Functional conservation testing:

• Express BBR orthologs from different species in rice BBR mutants

• Test complementation ability of distantly related BBR proteins

• Compare DNA binding specificities of BBR orthologs using in vitro assays

• Perform domain-swapping experiments to identify regions responsible for species-specific functions

Target gene conservation:

• Compare BBR binding sites across species using ChIP-seq

• Identify conserved versus species-specific targets

• Analyze evolutionary conservation of BBR binding motifs

• Determine if BBR regulates similar developmental processes across species

Correlation with morphological evolution:

• Analyze BBR sequence divergence in relation to leaf shape diversity

• Compare expression patterns of BBR across species with different leaf morphologies

• Investigate if BBR neo-functionalization or sub-functionalization events correlate with morphological innovations

• Use CRISPR to introduce specific BBR mutations that mimic evolutionary changes

This evolutionary perspective will provide insights into how BBR function has been maintained or altered throughout plant evolution, potentially revealing fundamental principles of developmental regulation.