Recombinant Arabidopsis thaliana Uncharacterized protein At3g49720 (At3g49720)

What structural features and functional domains are predicted for At3g49720?

Methodological Approach:

• Sequence Analysis: Retrieve coding sequences (e.g., NM_114832.4, NM_001339437.1) from the NCBI RefSeq database to identify open reading frames (ORFs) and splice variants .

• Transmembrane Prediction: Use tools like TMHMM or Phobius to predict transmembrane helices. At3g49720 contains 2–4 predicted α-helical transmembrane domains, suggesting a role in membrane-associated processes .

• Post-Translational Modifications: Perform in silico analysis with NetPhos or SignalP to identify phosphorylation sites or signal peptides. No canonical signal peptides are detected, implying intracellular membrane localization .

Data Table 1: At3g49720 Gene and Protein Features

How can researchers clone and express recombinant At3g49720 for functional studies?

Methodological Approach:

• ORF Cloning: Amplify the At3g49720 ORF (777 bp) using gene-specific primers designed with 5′ restriction sites for directional cloning into vectors like pcDNA3.1+/C-(K)DYK .

• Heterologous Expression: Express the protein in E. coli (BL21-DE3) or plant protoplasts. For membrane proteins, use detergent solubilization (e.g., DDM) and nickel-affinity chromatography for purification .

• Validation: Confirm expression via Western blot using anti-DYKDDDDK antibodies for tagged constructs .

What experimental strategies identify tissue-specific expression patterns of At3g49720?

Methodological Approach:

• Promoter-GUS Fusion: Generate transgenic Arabidopsis lines with the At3g49720 promoter driving β-glucuronidase (GUS). Stain tissues to visualize activity .

• RNA-Seq: Isolate nuclei from specific cell types using INTACT (Isolation of Nuclei TAgged in specific Cell Types) . Sequence RNA to quantify transcripts (e.g., 85 million reads per replicate, >75% mapping rate) .

How is subcellular localization determined for At3g49720?

Methodological Approach:

• Confocal Microscopy: Fuse At3g49720 with GFP under a constitutive promoter (e.g., 35S) and transiently express in Nicotiana benthamiana epidermal cells .

• Subcellular Fractionation: Separate membrane fractions via sucrose density gradient centrifugation. Validate with organelle-specific markers (e.g., plasma membrane ATPase, tonoplast TIP1) .

How do researchers resolve contradictions in At3g49720 localization data?

Methodological Approach:

• Comparative Assays: Repeat localization experiments across multiple systems (e.g., protoplasts, stable transgenics). For example, aequorin-tagged lines in Arabidopsis roots vs. leaves may show context-dependent localization .

• Knockout Complementation: Express GFP-tagged At3g49720 in at3g49720 T-DNA mutants. Compare localization with wild-type backgrounds to rule out overexpression artifacts .

What functional redundancy exists between At3g49720 and its paralogs (e.g., At5G65810)?

Methodological Approach:

• CRISPR-Cas9 Knockouts: Generate double/triple mutants of At3g49720 and paralogs. Phenotype under stress (e.g., pathogen exposure) using root growth assays .

• Transcriptomic Profiling: Compare RNA-seq data from mutants vs. wild-type to identify dysregulated genes (e.g., PDF1.2, WRKY33) .

Data Table 2: Phenotypic Analysis of At3g49720 Mutants

What signaling pathways involve At3g49720?

Methodological Approach:

• Calcium Imaging: Treat Aequorin-expressing plants with pathogen-associated molecular patterns (PAMPs). Measure cytosolic Ca²⁺ flux using luminescence assays .

• Pharmacological Inhibition: Apply Gd³⁺ (Ca²⁺ channel blocker) to test dependency of defense genes (e.g., MPK3) on Ca²⁺ signaling .

How are interaction partners of At3g49720 identified?

Methodological Approach:

• Yeast Two-Hybrid Screening: Screen a cDNA library using At3g49720 as bait. Validate hits with co-IP in plant extracts .

• Affinity Purification-MS: Express FLAG-tagged At3g49720 in transgenic lines. Immunoprecipitate complexes and identify proteins via LC-MS/MS .

What evolutionary insights exist for At3g49720 homologs?

Methodological Approach:

• Phylogenetic Analysis: Align At3g49720 with orthologs (e.g., Momordica charantia LOC111005582, Oryza sativa Os01g0144000) using MUSCLE. Construct trees with maximum likelihood (RAxML) .

• Synteny Mapping: Compare genomic regions across species (e.g., Amborella trichopoda) to infer ancestral gene duplications .

How are transcript isoforms of At3g49720 characterized?

Methodological Approach:

• RACE-PCR: Perform 5′/3′ rapid amplification of cDNA ends to identify alternative splice sites .

• Nanopore Sequencing: Use long-read RNA-seq to resolve full-length isoforms in pollen or meiocytes .