Functional Characterization of Thaumatin-like Protein 8 (TLP8) Using the CRISPR-Cas9 System
Bibliographic record
Abstract
Barley (Hordeum vulgare) and oat (Avena sativa) are important industrial cereals in Canada, notable for their substantial (1-3, 1-4)-β-D-glucan content, which is beneficial to human health. Thaumatin-like protein 8 (TLP8), a cysteine-rich protein in barley, has been shown to bind to (1- 3, 1-4)-β-D-glucan. Higher TLP8 expression in barley varieties has been linked to reduced (1-3, 1-4)-β-D-glucan content, potentially improving malting quality. However, the interaction between TLP8 and (1-3, 1-4)-β-D-glucan is not fully understood. We hypothesize that knocking out TLP8 could increase (1-3, 1-4)-β-D-glucan levels, a desirable trait for human consumption in both barley and oat. To investigate the function of TLP8 in these cereals, we employed the CRISPR-Cas9 gene-editing system. Constructs containing guide RNAs targeting the TLP8 gene were created and introduced into barley and oat tissues via Agrobacterium-mediated transformation and particle gun bombardment. In barley, immature embryos were inoculated with Agrobacterium, and after three rounds of hygromycin selection, three transgenic events were confirmed. Additionally, 200 barley calli pieces were bombarded, and following three rounds of hygromycin selection (20 mg/L), 14 plants were regenerated, with six confirmed as transgenic. Transformation efficiencies in barley were 30% and 43% using biolistics and Agrobacterium transformation, respectively.Bombardment of 100 oat calli resulted in the regeneration of 39 plants, of which 22 were confirmed as transgenic, yielding a transformation efficiency of 56%. These transgenic barley and oat plants were analysed further with Next Generation Sequencing to identify gene edits. Mutagenesis of the TLP8 region was confirmed, with one substitution being observed in barley, and numerous mutations observed in oat (editing frequency of 41%). Gene edits in oat included 1-bp, 4-bp or 12-bp indels near the PAM sequence of the TLP8 guide region. These mutants are being advanced to the next generation to obtain homozygous plants for phenotypic analysis. To further elucidate TLP8’s role, recombinant TLP8 proteins were expressed and purified from E. coli and assessed for β-glucanase activity using tetrazolium blue dye. These recombinant proteins exhibited significant β-glucanase activity on barley β-glucan. Homozygous CRISPR mutants will be subjected to additional studies to determine the role of TLP8 in β-glucan regulation and biosynthesis
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How this classification was reachedexpand
Full frame machine prediction
Teacher imitationNot calibrated prevalence, not ground truth. Human validation pending. The Gemma side is a direct model label for every work in the frame, read from the title-only record. The Codex side is a classifier learned from the 10,348 direct Codex labels and calibrated to design-weighted sample rates; fields without enough sample support carry no Codex call. Candidate is the union of the two sides; consensus is their intersection. These outputs are machine_predicted_unvalidated and are not human labels.
Distilled classifier scores by category (both heads)
| 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 | 0.001 |
| Insufficient payload (model declined to judge) | 0.001 | 0.001 |
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 source (direct Gemma or distilled Codex), 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".