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首页> 外文期刊>Journal of insect biotechnology and sericology >CRISPR/Cas9-mediated somatic mutation of the sex-linked translucent (os) genein the silkworm, Bombyx mori
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CRISPR/Cas9-mediated somatic mutation of the sex-linked translucent (os) genein the silkworm, Bombyx mori

机译：Crispr / Cas9介导的性联系半透明（OS）Genein的体细胞突变，蚕丝虫，Bombyx Mori

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The Z chromosome-linked sex-linked translucent (os) mutation in the silkworm Bombyx morl causes a slightly translucent larval integument that results from the reduction of urate granules in epidermal cells. We previously concluded that mutations in anamino 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 mosaiclarvae 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 isrequired 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 inall lepidopteran insects. On the other hand, OS proteins were not found in some insect species, such as Apis melllfera, Drosophila melanogaster, and Tribollum castane-um. It is considered that os genes play an important role in lepidopteran insects for their survival in nature.
机译：Z染色体联系的性状联系半透明（OS）突变在家蚕BOMBYX MORL中导致略微半透明的幼虫物质，从表皮细胞中的尿液颗粒的减少产生。我们以前得出结论，属于溶质载体家族的Anamino酸转运蛋白基因的突变对OS突变表型通过独立OS突变菌株的位置克隆和序列分析负责。然而，失踪了使用功能分析的直接证据。因此，我们使用CRISPR / CAS9系统对OS候选基因进行了体制型马赛克分析。将体外合成的单引导RNA（SGRNA）和Cas9蛋白混合并注入P50T菌株的卵中。我们成功地获得了Mosaiclarvae，其中表皮包括白色和略微半透明的区域。在从G0成年腿提取的基因组DNA中诱导各种突变在OS基因的靶位点，表明氨基酸转运蛋白因表皮细胞颗粒中的尿酸充分积累。此外，我们在24种Genome测序的鳞翅目和12个其他昆虫中搜索了OS同源蛋白质。系统发育分析表明，OS蛋白是保守的鳞翅目昆虫。另一方面，在某些昆虫物种中未发现OS蛋白，例如Apis Melllfera，果蝇黑色糖蛋白酶和Tribollum Castane-Um。认为OS基因在鳞翅目昆虫中发挥着重要作用，以便在自然中存活。

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