CRISPR/Cas9-mediated somatic mutation of the sex-linked translucent (os) gene in the silkworm, Bombyx mori
CRISPR/Cas9-mediated somatic mutation of the sex-linked translucent (os) gene in the silkworm, Bombyx mori
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DOI：
10.11416/jibs.88.2_031
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发表时间：
2019
影响因子：
--
通讯作者：
Kenta Tomihara;Katsuya Satta;T. Shimada;T. Kiuchi
中科院分区：
文献类型：
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作者：
Kenta Tomihara;Katsuya Satta;T. Shimada;T. Kiuchi
家蚕Z染色体伴性连锁半透明突变导致幼虫体壁微透明，这是由于表皮细胞中尿酸盐颗粒减少所致。我们以前的结论是，在一个氨基酸转运蛋白基因属于溶质载体家族的突变是负责独立的os突变株的定位克隆和序列分析的os突变表型。然而，使用功能分析的直接证据缺失。因此，我们使用CRISPR/Cas9系统对os候选基因进行了体细胞嵌合体分析。将体外合成的单向导RNA（sgRNA）和Cas9蛋白混合并注射到p50 T菌株的卵中。我们成功地获得了镶嵌幼虫，其中表皮包括白色和轻微半透明的区域。从G 0成年人腿提取的基因组DNA中的os基因的靶位点处已被诱导发生各种突变，表明氨基酸转运体基因是尿酸在表皮细胞颗粒中充分积累所必需的。此外，我们还在24种基因组测序的鳞翅目昆虫和12种其他昆虫中搜索了OS同源蛋白。系统进化分析表明OS蛋白在所有鳞翅目昆虫中是保守的。另一方面，OS蛋白在一些昆虫物种中未被发现，如意大利蜜蜂、黑腹果蝇和赤拟谷盗。os基因在鳞翅目昆虫的生存中起着重要的作用。
The Z chromosome-linked sex-linked translucent (os) mutation in the silkworm Bombyx mori causes a slightly translucent larval integument that results from the reduction of urate granules in epidermal cells. We previously concluded that mutations in an amino acid transporter gene belonging to the solute carrier family are responsible for the os mutant phenotype by positional cloning and sequence analysis of independent os mutant strains. However, direct evidence using functional analysis was missing. Therefore, we performed somatic mosaic analysis of the os candidate gene using the CRISPR/Cas9 system. An in vitro synthesized single guide RNA (sgRNA) and Cas9 protein were mixed and injected into eggs of the p50T strain. We successfully obtained mosaic larvae in which the epidermis comprised both white and slightly translucent areas. Various mutations had been induced at the target sites of the os gene in the genomic DNA extracted from G0 adult legs, indicating that the amino acid transporter gene is required for the sufficient accumulation of uric acid in epidermal cells granules. In addition, we searched OS homologous proteins in 24 genome-sequenced lepidopteran and 12 other insects. Phylogenetic analysis showed that the OS protein is conserved in all lepidopteran insects. On the other hand, OS proteins were not found in some insect species, such as Apis mellifera, Drosophila melanogaster, and Tribolium castaneum. It is considered that os genes play an important role in lepidopteran insects for their survival in nature.