昆虫学报2021，Vol.64Issue(12)：1367-1376,10.DOI:10.16380/j.kcxb.2021.12.002
敲除ebony基因导致二化螟体表黑色素沉积
Knockout of ebony gene leads to melanin pigmentation in the rice stem borer,Chilo suppressalis(Lepidoptera:Crambidae)
摘要
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 2586 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.关键词
二化螟/基因编辑/CRISPR/Cas9/ebony/标记基因/表型/脱靶效应Key words
Chilo suppressalis/gene editing/CRISPR/Cas9/ebony/marker gene/phenotype/off-target effect分类
生物科学引用本文复制引用
孙浩,黄镜梅,刘艳,葛文超,王帅,杨凤霞,高聪芬,吴顺凡..敲除ebony基因导致二化螟体表黑色素沉积[J].昆虫学报,2021,64(12):1367-1376,10.基金项目
国家自然科学基金优秀青年科学基金项目(32022011) （32022011）
江苏省农业科技自主创新资金项目(CX[19]3003) （CX[19]3003）
