关键词: 二化螟, 基因编辑, CRISPR/Cas9, ebony, 标记基因, 表型, 脱靶效应

Abstract:

【Aim】 The rice stem borer, Chilo suppressalis, is a destructive rice pest in China and other Asian countries. However, due to lack of genetic tools, the functional genomic studies in C. suppressalis are seriously constrained. The aim of the study is to use a marker gene, ebony, to establish a gene editing system based on CRISPR/Cas9 technology in C. suppressalis. 【Methods】 With the amino acid sequences of Bombyx mori ebony protein as a query, the putative C. suppressalis ebony gene was obtained on its genomic database by the TBLASTN program. The full-length cDNA of ebony gene of C. suppressalis was cloned by PCR and subjected to bioinformatical analysis. The expression patterns of Csebony at different developmental stages (egg, larval, pre-pupal, pupal, and male and female adult stages) and in multiple tissues (head, epidermis, fat body, gut, and Malpighian tubules) of the 4th instar larvae of C. suppressalis were analyzed by qRT-PCR. Finally, we performed targeted knockout of ebony gene in C. suppressalis by microinjecting the ribonucleoprotein complexes specific guide RNA/Cas9 protein into the newly laid eggs within 2 h based on the CRISPR/Cas9 gene editing technology. 【Results】 The full-length cDNA of Csebony gene (GenBank accession no.: MZ846208) of C. suppressalis was cloned. It contains a 2 586 bp ORF encoding 861 amino acids, with the molecular mass of 9.5 kD and theoretical isoelectric point of 5.10. Csebony has no signal peptide sequence at the N-terminus. Domain analysis showed that Csebony has three conserved domains. Phylogenetic analysis indicated that Csebony is most closely related to Ostrinia furnacalis ebony. The qRT-PCR results showed that Csebony was highly expressed in the pupal stage and head. Knockout of Csebony caused melanin pigmentation in larvae, pupae, and adults of C. suppressalis. 【Conclusion】 The results showed that Csebony is involved in regulating cuticle pigmentation of C. suppressalis, and CRISPR/Cas9-based genome editing technology is effective in C. suppressalis. We can use visible marker gene to establish CRISPR/Cas9-based genome editing system in non-model organisms, so as to offer a valuable genetic tool for the study of functional genomics in C. suppressalis.

Key words: Chilo suppressalis, gene editing, CRISPR/Cas9; ebony, marker gene, phenotype, off-target effect