Expression of a Wheat Endosperm 14-3-3 Protein and Its Interactions with Starch Biosynthetic Enzymes in Amyloplasts
Bibliographic record
Abstract
Wheat endosperm starch is the major determinant of grain yield and processing quality.The quality and quantity of starch are controlled by a number of starch biosynthetic enzymes.14-3-3 proteins,involved in many biological processes,are ubiquitous and important regulators in all eukaryotic cells from yeast to mammals and plants.Protein-protein interactions between a wheat endosperm 14-3-3 protein and starch biosynthetic enzymes from amyloplast were investigated in this study.A 14-3-3 gene was cloned from developing wheat endosperm and inserted into plasmid vectors pET29c and pET41c.The recombinant vectors were transformed into Escherichia coli strain BL21-CodonPlus(DE3)-RP and expressed at very high level.The fusion protein existed mainly as an insoluble inclusion body after extraction by BugBuster Protein Extraction Reagent.The soluble fusion protein was purified by bounding to S-protein agarose,while the inclusion body should be dissolved in 8 mol L- 1urea and refolded firstly.Sucrose synthase activity was observed to be inhibited by exogenous recombinant 14-3-3 protein in a dosage-dependent manner,which suggested the refolded protein was successfully activated and could be used in the following research.The purified recombinant 14-3-3 protein was bound to S-protein agarose as a biochemical bait,and then incubated with wheat amyloplast extract.Proteins interacting specifically with the 14-3-3 protein and remaining on the resin were analyzed by SDS-PAGE and Western blot.These assays showed that starch synthase I(SSI),starch synthase II(SSII),starch branching enzyme IIa(SBEIIa),starch branching enzyme IIb(SBEIIb),and ADP glucose pyrophosphorylase large subunit(SH2)interacted with 14-3-3 protein,whereasSBEI,starch phosphorylase(SP),D-enzyme(DE),and ADP glucose pyrophosphorylase small subunit(BT2)did not bind with the 14-3-3 protein.The results suggest a role for the wheat endosperm 14-3-3 protein in regulation of grain starch biosynthetic enzymes.
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How this classification was reachedexpand
Full frame distilled prediction
Teacher imitationNot calibrated prevalence, not ground truth. Human validation pending. Learned from the 10,348 direct Codex labels and 10,348 direct Gemma labels. Candidate is the union of thresholded teacher heads; consensus is their intersection. These outputs are machine_predicted_unvalidated and are not human labels or direct frontier model labels.
Codex and Gemma teacher scores by category
| Category | Codex | Gemma |
|---|---|---|
| Metaresearch | 0.000 | 0.000 |
| Meta-epidemiology (narrow) | 0.000 | 0.000 |
| Meta-epidemiology (broad) | 0.000 | 0.000 |
| Bibliometrics | 0.000 | 0.000 |
| Science and technology studies | 0.000 | 0.000 |
| Scholarly communication | 0.000 | 0.000 |
| Open science | 0.000 | 0.000 |
| Research integrity | 0.000 | 0.000 |
| Insufficient payload (model declined to judge) | 0.000 | 0.000 |
Machine scores (provisional)
The two teacher heads of the student model, read on this work. A score orders the frame for review; it never asserts a category, and the validation status ships verbatim with every row.
Baseline scores from an immature model (maturity gate not passed, 7 training rounds). Scores rank; they never assert a category.
score_only:v0-immature-baseline · verbatim from the scoring run: score_only means the number may rank works, and no category label ships from itClassification
machine, unvalidatedMachine predicted; a candidate call from one teacher head, not a consensus.
How this classification was reached, model by model and score by score, is at the end of the page under "How this classification was reached".