Recombinant Ipomoea purpurea ATP synthase subunit b, chloroplastic (atpF)

What molecular techniques are recommended for isolating the atpF gene from Ipomoea purpurea?

For isolating the atpF gene from I. purpurea, a combination of RNA extraction, reverse transcription, and PCR amplification is recommended. Start by extracting total RNA using Trizol reagent from fresh leaf tissue. First-strand cDNA synthesis should be performed using M-MLV reverse transcriptase with oligo(dT) primers (incubation at 37°C for 50 min, followed by heating at 70°C to terminate the reaction) . For PCR amplification, design specific primers based on conserved regions of atpF sequences from related Ipomoea species. The PCR reaction can be performed using a high-fidelity DNA polymerase such as KOD-Plus-Neo with the following conditions: pre-denaturation at 94°C for 2 min, followed by 30 cycles of denaturation at 98°C for 10 s, annealing at 54-56°C for 30 s, extension at 68°C (1 min per kb), and final extension at 68°C for 10 min .

What are the optimal expression systems for producing recombinant Ipomoea purpurea atpF protein?

The optimal expression system for recombinant I. purpurea atpF likely resembles that used for other chloroplast genes in Ipomoea species. Based on research with related ATP synthase genes, E. coli BL21(DE3) is recommended as an expression host, using vectors such as pET32a(+) that provide an N-terminal fusion tag (like thioredoxin) to enhance solubility . The expression construct should be designed using sequence and ligation independent cloning (SLIC) methods for efficient assembly. For expression induction, IPTG concentrations between 0.1-2.0 mM can be tested, although as observed with similar genes, there may not be significant differences in expression levels across this concentration range . Expression at lower temperatures (18°C for 16 hours) rather than standard 37°C conditions will likely improve the yield of properly folded protein .

How can researchers differentiate between native and recombinant atpF protein in experimental analyses?

To differentiate between native and recombinant atpF protein:

• Incorporate an affinity tag (His-tag, GST, or TRX) at the N- or C-terminus of the recombinant protein to enable:

  • Selective purification using affinity chromatography

  • Immunodetection using tag-specific antibodies on Western blots

• Develop specific antibodies against unique epitopes in the atpF protein if planning extensive studies

• Use mass spectrometry to:

  • Verify the identity of the recombinant protein

  • Confirm post-translational modifications that may differ between native and recombinant forms

  • Calculate the exact molecular weight (native atpF vs. recombinant fusion protein)

• Perform activity assays comparing enzymatic properties between native chloroplast extracts and purified recombinant protein

What are the potential evolutionary implications of sequence variations in atpF across different Ipomoea species?

Sequence variations in atpF across Ipomoea species can provide valuable insights into evolutionary relationships and adaptation mechanisms. Analysis of chloroplast genomes from various Ipomoea species has revealed multiple polymorphic sites and variable regions that serve as nucleotide hotspots . The LSC region, where atpF is located, contains some of these variable regions. These variations can be used to infer phylogenetic relationships among Ipomoea species.

Codon usage bias analysis of chloroplast genes in Ipomoea has shown that most genes have a preference for T over A and G over C in the third position . For ATP synthase genes specifically, the usage frequency patterns may reflect selective pressures related to functional constraints. Comparative analysis of atpF sequences could reveal whether this gene is under purifying selection (conservation of function) or has accumulated adaptive mutations across different Ipomoea species in different environments.

What is the recommended protocol for cloning the atpF gene from Ipomoea purpurea into an expression vector?

The recommended protocol for cloning atpF from I. purpurea into an expression vector involves:

• RNA Isolation and cDNA Synthesis:

  • Extract total RNA from young leaves using TRIZOL reagent

  • Synthesize first-strand cDNA using M-MLV reverse transcriptase with oligo(dT) primers

  • Incubate at 37°C for 2 minutes, add reverse transcriptase, continue at 37°C for 50 minutes, then heat at 70°C to terminate

• PCR Amplification:

  • Design primers based on conserved regions of atpF from related species

  • Add vector-compatible overhangs to facilitate cloning

  • Perform PCR using high-fidelity DNA polymerase under optimized conditions

• Vector Preparation and Cloning:

  • Amplify the expression vector (e.g., pET32a+) with primers containing overhangs complementary to the atpF gene

  • Use sequence and ligation independent cloning (SLIC):

    • Treat both PCR products with T4 DNA polymerase for 30 seconds at 37°C

    • Mix and anneal at 75°C for 10 minutes, cooling naturally to room temperature

    • Transform directly into E. coli competent cells

• Verification:

  • Confirm recombinants by colony PCR

  • Verify by restriction digestion (e.g., with XbaI)

  • Confirm by DNA sequencing

What methods are most effective for analyzing differential expression of atpF in Ipomoea purpurea tissues?

For analyzing differential expression of atpF across I. purpurea tissues, a combination of the following methods is most effective:

• RNA-Seq Analysis:

  • Perform whole-transcriptome sequencing from different tissues

  • Map reads to the assembled transcriptome reference

  • Quantify expression levels using metrics like FPKM or TPM

  • This approach can identify tissue-specific expression patterns and potential splice variants

• Semi-quantitative RT-PCR:

  • Design primers specific to atpF and a housekeeping gene (e.g., GAPDH)

  • Extract RNA from different tissues (stems, mature leaves, young leaves, roots)

  • Perform RT-PCR with consistent cycle numbers

  • Analyze band intensity to compare relative expression levels

• Quantitative Real-time PCR (qRT-PCR):

  • Design primers specific to the atpF gene with amplicon length of 100-200 bp

  • Use multiple reference genes for normalization

  • Calculate relative expression using the 2^-ΔΔCt method

  • Perform statistical analysis to validate significance of expression differences

• Digital Gene Expression (DGE) Profiling:

  • Generate short sequence tags from mRNAs

  • Map tags to reference sequences

  • Count tag frequencies to quantify expression levels

How can researchers interpret polymorphic sites in the atpF gene across Ipomoea species?

Researchers can interpret polymorphic sites in the atpF gene using the following approaches:

• Sequence Alignment and Variant Detection:

  • Align atpF sequences from multiple Ipomoea species

  • Identify singleton variable sites (occurring in only one species) and parsimony informative sites (occurring in multiple species)

  • Classify variants by type (e.g., two, three, or four variants at a single position)

• Nucleotide Diversity Analysis:

  • Calculate nucleotide diversity (Pi) across the aligned sequences

  • Identify hotspot regions with elevated diversity (Pi ≥ 0.015)

  • Compare diversity patterns with other chloroplast genes

• Selection Pressure Analysis:

  • Calculate the ratio of non-synonymous to synonymous substitutions (dN/dS)

  • Determine if the gene is under purifying selection (dN/dS < 1), neutral evolution (dN/dS ≈ 1), or positive selection (dN/dS > 1)

  • Map selection signals to specific functional domains of the protein

• Phylogenetic Analysis:

  • Construct phylogenetic trees based on atpF sequences

  • Compare with trees constructed using other chloroplast genes

  • Identify instances of potential horizontal gene transfer or introgression

What are the key considerations for optimizing recombinant atpF protein expression in E. coli?

Key considerations for optimizing recombinant atpF protein expression in E. coli include:

Additional optimization may involve testing different fusion tags, optimizing the linker sequence between the tag and atpF, and screening multiple colonies for expression variation.

How can structural predictions inform functional studies of recombinant atpF protein?

Structural predictions can significantly inform functional studies of recombinant atpF protein through:

• Homology Modeling:

  • Generate 3D models based on solved structures of homologous ATP synthase subunit b proteins

  • Identify conserved structural motifs essential for function

  • Predict the location and structure of transmembrane domains

• Identification of Functional Domains:

  • Map regions involved in protein-protein interactions within the ATP synthase complex

  • Identify residues critical for proton translocation

  • Predict potential sites for post-translational modifications

• Rational Design of Mutants:

  • Select residues for site-directed mutagenesis based on structural predictions

  • Design truncated variants to study domain-specific functions

  • Create chimeric proteins with domains from different species to investigate evolutionary adaptations

• Protein-Protein Interaction Studies:

  • Predict interaction surfaces with other ATP synthase subunits

  • Design co-expression experiments with partner proteins

  • Guide the development of pull-down assays to verify predicted interactions

What are the remaining knowledge gaps in understanding recombinant Ipomoea purpurea atpF?

Despite advances in understanding chloroplast genomes of Ipomoea species, significant knowledge gaps remain regarding I. purpurea atpF:

• Species-specific sequence data: Complete sequence characterization of atpF specifically from I. purpurea is still needed, as current knowledge is largely extrapolated from related species .

• Structure-function relationships: The specific structural features that distinguish I. purpurea atpF from other species and their functional implications remain unexplored.

• Post-translational modifications: The types and patterns of post-translational modifications on native atpF in I. purpurea chloroplasts and their impact on protein function are unknown.

• Environmental response patterns: How atpF expression and function respond to various environmental stresses (particularly herbicide exposure) in I. purpurea needs further investigation, especially given the species' known herbicide resistance mechanisms .

• Protein-protein interaction network: The complete interaction map of atpF with other subunits of the ATP synthase complex and potentially with other proteins in I. purpurea chloroplasts remains to be elucidated.

How might future technological advances enhance research on recombinant Ipomoea purpurea atpF?

Future technological advances that could enhance research on recombinant I. purpurea atpF include: