Identification of early flowering mutant gene in Phalaenopsis amabilis (L.) Blume for sgRNA construction in CRISPR/Cas9 genome editing system

• Main Authors: Suputri, N.P.A.E.O., Prasojo, I.S., Prabowo, L.A.T., Purwestri, Y.A., Purnomo, Purnomo, Semiarti, E.
• Format: Article PeerReviewed
• Published: 2024
• Subjects: Molecular Evolution; Plant Biology; Biological Sciences
• Online Access: https://repository.ugm.ac.id/279106/; https://www.scopus.com/inward/record.uri?eid=2-s2.0-85161166984&doi=10.1590%2f1519-6984.268133&partnerID=40&md5=6a26dc134c63748e6d20a6063a93fb79
• Institution: Universitas Gadjah Mada

Phalaenopsis amabilis (L.) Blume commonly called Moth Orchid (Orchidaceae) is a natural orchid species designated as the National Flower of Indonesia for its beautiful flower shape and long-lasting flowering period. Basically, P. amabilis has a long vegetative phase that cause late flowering, about 2 to 3 years for flowering, hence a method to shorten vegetative period is desired. The latest technological approach that can be used to accelerate flowering of P. amabilis is the CRISPR/Cas9 genome editing method to inactivate the GAI (Gibberellic Acid Insensitive) gene as a mutant gene that can accelerate the regulation of FLOWERING TIME (FT) genes flowering biosynthesis pathway. The approach that needs to be taken is to silence the GAI gene with a knockout system which begins with identifying and characterizing the GAI target gene in the P. amabilis which will be used as a single guide RNA. CRISPR/Cas9 mediated knockout efficiency is highly dependent on the properties of the sgRNA used. SgRNA consists of a target sequence, determining its specificity performance. We executed phylogenetic clustering for the PaGAI protein with closely related orchid species such as Dendrobium capra, Dendrobium cultivars and Cymbidium sinensis. SWISS-Model as tool webserver for protein structure homology modeling. Results show that P. amabilis has a specific domain with the occurrence of point mutations in the two conservative domains. Therefore, a single guide RNA reconstruction needs to be implemented. © 2024, Instituto Internacional de Ecologia. All rights reserved.