Creation of hypoallergenic mustard (Brassica juncea) through genome editing and development of precise base editing tools for plants

Publikation: Qualifikations-/Studienabschlussarbeit › Dissertation

Autoren

  • Dingbo Zhang

Organisationseinheiten

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Details

OriginalspracheEnglisch
QualifikationDoctor rerum naturalium
Gradverleihende Hochschule
Betreut von
  • Jens Boch, Betreuer*in
Förderer
  • Bundesministerium für Bildung und Forschung (BMBF)
Datum der Verleihung des Grades31 Aug. 2023
ErscheinungsortHannover
PublikationsstatusVeröffentlicht - 2023

Abstract

Eine verbesserte landwirtschaftliche Sicherheit durch neue Züchtungsverfahren ist dringend erforderlich, um den Zugang zu nahrhaften Lebensmitteln weltweit zu verbessern. Das Genom-Engineering mit Hilfe von CRISPR (clustered regularly interspaced short palindromic repeats)-Technologien oder TALE (transcription activator-like effector)-Technologien bietet die einzigartige Möglichkeit, gezielt Gene für eine präzise Züchtung zu verändern. Diese Technologie ist vielversprechend für verschiedene Anwendungen in der Allergieforschung. Das Hauptallergen Bra j I aus braunem Senf (Brassica juncea) ist ein Saatgut-Speicherprotein, das zur 2S-Albuminfamilie gehört. Ein Ziel dieser Arbeit war die Schaffung einer hypoallergenen Senfsorte durch den Einsatz von Genome Editing-Techniken und ein zweites Ziel war die Entwicklung neuer Baseneditoren für Pflanzen. Zunächst wurden zwei CRISPR/Cas9-Konstrukte mit Multiplex-Single-Guide-RNAs eingesetzt, um große Deletionen oder Frameshift-Mutationen in den beiden Homöologen Bra j IA und Bra j IB in zwei Linien des braunen Senf (Terratop und CR2664) zu induzieren. In den transgenen T0-Senfpflanzen wurden hohe Mutationseffizienzen beobachtet. Das Bra j IB-Allel wies in vier Linien große Deletionen zwischen 566 und 790 bp auf. Außerdem wiesen neun von 18 Terratop-T0-Linien kleine Indels in den Zielregionen auf. In ähnlicher Weise wiesen 14 der 16 analysierten CR2664 T0-Linien Indels auf, während drei Linien Mutationen in allen vier Bra j I-Allelen aufwiesen. Die Mutationen wurden stabil an die T1-Nachkommen vererbt. Darüber hinaus zeigten Immunoblotting-Ergebnisse eine Abnahme oder ein vollständiges Fehlen des Bra j I-Proteins in den Samenextrakten ausgewählter T1-Linien. Diese Arbeit unterstreicht den Wert von Genom Editing Technologien für die Schaffung hypoallergener Lebensmittelpflanzen. Zweitens wurden zwei Baseneditoren entwickelt: TALE-abgeleitete DddA-basierte Cytosin-Baseneditoren (TALE-DdCBEs) und TALE-abgeleitete Adenin-Baseneditoren (TALE-ABEs). Sie wurden entwickelt, um eine präzise Bearbeitung von C•G-to-T•A bzw. A•T-to-G•C zu erstellen. TALE-DdCBEs, die DddA-Varianten (DddA6 oder DddA11) enthielten, zeigten eine deutliche Verbesserung der Editierungseffizienz sowohl in Nicotiana benthamiana als auch in Reisprotoplasten. TALE-DdCBEs mit DddA11 wiesen eine bessere Sequenzkompatibilität für die Bearbeitung von Nicht-TC-Zielen auf. Darüber hinaus wurden verschiedene TALE-ABEs mit unterschiedlichen Desaminase-Fusionsarchitekturen in Reis und N. benthamiana getestet. Die Ergebnisse zeigten, dass TALE-ABEs A•T-to-G•C Umwandlungen im Reisprotoplasten ermöglichen. TALE-Base-Editoren können für nukleare Gene eingesetzt werden oder alternativ über N-terminale Targeting-Sequenzen die Genome von Plastiden oder Mitochondrien zum Ziel haben.

Zitieren

Creation of hypoallergenic mustard (Brassica juncea) through genome editing and development of precise base editing tools for plants. / Zhang, Dingbo.
Hannover, 2023. 127 S.

Publikation: Qualifikations-/Studienabschlussarbeit › Dissertation

Download
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