Biotechnological modulation of hordein accumulation in barley grains for molecular farming

verfasst von

Pouneh Pouramini

betreut von

Traud Winkelmann

Abstract

Producing high-quality recombinant proteins in plants is an excellent alternative to bacterial systems, yeasts, insects, or mammalian cells. However, the accumulation of proteins within a cell is spatially limited by the available energy resources and limitations of the biological building blocks. To overcome these hurdles, this work aims to achieve possibilities for increasing the protein yield with the help of two innovative technologies. To provide space and building blocks for the production of the recombinant protein in the barley grain, CRISPR/Cas9 technology was used to mutate barley storage protein genes, so-called B-Hordeins specifically. Since these storage proteins are essential in germination, grains with a low B-Hordein content will germinate more poorly. However, since these proteins are necessary for seed multiplication, the B-hordein mutants must be complemented with a temperature-controlled mechanism (lt-degron technology). For this purpose, a sequence was added N-terminally to the B-Hordein protein, which directs the corresponding coupled B-Hordein protein to degrade when the temperature increases. This means that under normal growth conditions, the B-Hordein is active and can support germination. When the high-value protein is to be formed, the plants are exposed to a higher temperature. To test this novel technology, wildtype and B-hordein mutant plants were transformed with two constructs for the endosperm-specific expression of the human Epidermal Growth Factor (EGF) and the human Fibroblast Growth Factor basic (FGFb) genes. ORF Genetics Iceland used the same constructs to produce these recombinant proteins commercially. After determining the genetic B-Hordein constitution of the barley cultivar Golden Promise used for transformation, several constructs were created for targeted mutagenesis of B-Hordein and several independent mutants were obtained. The grains of these segregating populations were characterised by reduced width, length and weight compared to the wild type. As expected, B-hordein M1 mutants had lower protein content and poorer germination. These characteristics were even more pronounced in the next homozygous generation. To gain a deeper insight into the morphological features of the B-hordein mutant grains, some progeny were examined in collaboration with the Research Centre Jülich using an automated seed phenotyping system. The results corroborated the visual observations. In collaboration with ORF Genetics in Iceland, the EGF and FGFb content of T1 grains was quantified. The results indicate that more recombinant protein can be obtained by reducing barley's storage proteins. A GFP- and B-Hordein-coupled fusion protein were transformed into Golden Promise to establish lt-degron-controlled protein degradation in barley. Initial studies on the temperature-controlled degradation of GFP show promising results. Compared to the control, which has a higher accumulation of GFP under temperature elevation, calli with an lt-degron-GFP T-DNA have a lower fluorescence after treatment at 37°C for 24 hours. In summary, it can be stated that improved recombinant protein accumulation in barley plants with a lower storage protein content appears possible. However, it remains to be tested whether the poorer germination capacity of B-hordein M2 mutants can be overcome by complementation with a construct for endosperm-specific accumulation of lt-degron-B-Hordein.

Organisationseinheit(en)

Abteilung Reproduktion und Entwicklung

Typ

Dissertation

Anzahl der Seiten

106

Publikationsdatum

12.09.2024

Publikationsstatus

Veröffentlicht

Elektronische Version(en)

https://doi.org/10.15488/17975 (Zugang: Offen)