关键词: 鳞翅目, 家蚕, 泌丝昆虫, 驯化基因, 转录因子, CRISPR/Cas9

Abstract: 【Aim】This study aims to explore the molecular evolution pattern of the putative domestication gene, the transcription factor ZnF-706 in different lepidopteran groups and in the domestication process of the silkworm Bombyx mori, and to explore its function in the silkworm by knockout of ZnF-706 via the CRISPR/Cas9 genome editing system.【Methods】 We analyzed the gene structure of ZnF-706 and investigated its tissue expression pattern in the day-3 5th instar larvae of the silkworm based on the published silkworm microarray data. Using phylogenetic analysis by Maximum Likelihood (PAML), we tested the evolution pattern of ZnF-706 in Lepidoptera. Based on the published genetic polymorphism data of the B. mori and Bombyx mandarina populations, we screened the artificial selection signal of ZnF-706 genic region. After analyzing the upstream 2 kb region of this gene, we detected the fixed SNPs in the domestic silkworm and predicted transcription factor binding motif in the region covering these SNPs. By knockout of this gene using CRISPR/Cas9 genome editing, we obtained the homozygosis mutant. The variation of cocoon weight and pupal weight of mutants was detected by using wild-type silkworm as the control. 【Results】 The encoded protein of ZnF-706 has a typical zinc finger protein domain. ZnF-706 is ubiquitously expressed in the silkworm larva, especially highly expressed in the cuticle, fat body and gonads. It evolved rapidly in three clades, namely Lepidoptera, Bombycoidea and Antherea yamamai. Strong artificial selection signals in the region covering this gene were detected, with significantly elevated population divergence (Fst) between the B. mori and B. mandarina populations and decreased population diversity (π) in the B. mori population, suggesting that it is located in a selective sweep. Nine fixed SNPs in the regulatory region of upstream of this gene were detected, and they were located in the motifs with transcription factor binding activity. Homozygous loss-of-function mutants of ΔZnF-706 showed weaker viability and significantly lower cocoon weight and pupal weight, as compared with the wild-type silkworm. But unlike in Drosophila melanogaster, the homozygous mutants of ZnF-706 in B. mori were not lethal.【Conclusion】 ZnF-706 evolves rapidly in Lepidoptera, especially silk producing insects, and is also under artificial selection during silkworm domestication, suggesting that it might be associated with cocoon silk. It may affect silk cocoon traits by direct regulation of silk protein gene, or by indirect regulation through affecting the growth and development of the silkworm. This study provides not only unique evidence from insect domestication for exploring the mechanism of artificial selection, but also cues to the further research on the transcriptional regulation of economic traits of the silkworm.

Key words: Lepidoptera, Bombyx mori; silk-producing insect, domestication gene, transcriptional factor, CRISPR/Cas9