Cloning and Functional Verification of Endogenous U6 Promoters for the Establishment of Efficient CRISPR/Cas9-Based Genome Editing in Castor (Ricinus communis)

Author:

Kanai Masatake1,Hikino Kazumi1,Mano Shoji12

Affiliation:

1. Laboratory of Organelle Regulation, National Institute for Basic Biology, Okazaki 444-8585, Japan

2. Basic Biology Program, Graduate Institute for Advanced Studies, The Graduate University for Advanced Studies, SOKENDAI, Okazaki 444-8585, Japan

Abstract

Castor (Ricinus communis) seeds are rich in a type of hydroxy fatty acid called ricinoleic acid, which is in high demand for the production of plant-based plastics, lubricants, and hydraulic oils. However, the high content of ricin, a toxic protein, in these seeds has restricted further expansion in the area of castor cultivation. Therefore, the development of ricin-free castor is needed. Genome editing technology, although successfully applied in several plant species, is still in the developing stages in castor and awaits the identification of an endogenous U6 promoter with robust function. Here, we searched for U6 small nuclear RNA (snRNA) genes in the castor genome. This led to the identification of six U6 snRNA genes. The promoters of these U6 snRNA genes were cloned, and their function was examined in castor cells using the particle delivery method. The results showed that a U6 promoter length of approximately 300 bp from the transcription start site was sufficient to activate gene expression. This study provides insights into the endogenous castor U6 promoter sequences and outlines a method for verifying the function of U6 promoters in plants using the particle delivery system.

Funder

JSPS KAKENHI

Publisher

MDPI AG

Subject

Genetics (clinical),Genetics

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