CRISPR/Cas9 mediated BLOS2 knockout resulting in disappearance of yellow strips and white spots on the larval integument in Spodoptera litura
CRISPR/Cas9 mediated BLOS2 knockout resulting in disappearance of yellow strips and white spots on the larval integument in Spodoptera litura
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CRISPR/Cas9介导的BLOS2敲除导致斜纹夜蛾幼虫体皮上的黄色条带和白点消失
DOI：
10.1016/j.jinsphys.2017.09.008
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发表时间：
2017
影响因子：
2.2
通讯作者：
Dong Shuang Lin
中科院分区：
文献类型：
--
作者：
Zhu Guan Heng;Peng Ying Chuan;Zheng Mei Yan;Zhang Xiao Qing;Sun Jia Bin;Huang Yongping;Dong Shuang Lin
定制设计的细菌CRISPR/Cas9系统最近已在一些昆虫中使用，这表明了基因功能和转基因昆虫研究的强大技术。然而，其在鳞翅目害虫中的使用很少。本文报道了利用CRISPR/Cas9系统对斜纹夜蛾（Spodoptera litura）溶酶体相关细胞器复合体1亚基2（BLOS 2）基因的生物合成进行突变，并通过对突变体的aS分析鉴定了SlitBLOS 2基因片段。通过本地基本的BLAST技术从lituratranscriptome数据库中克隆lituratranscriptome基因，并通过RACE策略获得lituratranscriptome基因的全长cDNA。为了阐明SlitBL 0 S2的功能，实现了基于CRISPR/Cas9的SlitBL 0 S2的靶向突变，在62.3-70.6%的G 0代幼虫中显示出镶嵌半透明的体壁。进一步的PCR基因型分析表明，在SlitBLOS 2特异性靶位点发生了多种突变。在G1代获得了一个纯合子突变体，其幼虫体壁上的黄色条纹和白色斑点完全消失。我们的研究清楚地证明了SlitBLOS 2在珠被着色中的功能，从而为基于基因组编辑的斜纹夜蛾以及其他鳞翅目害虫的基因功能研究和害虫防治策略提供了有用的标记基因。
The custom-design bacterial CRISPR/Cas9 system has been recently used in some insects, indicating a powerful technique for studies on gene function and transgenic insects. However, its use in lepidopteran pests is scarce. Here, we reported a CRISPR/Cas9 system mediated mutagenesis ofbiogenesis of lysosome-related organelles complex1, subunit 2(BLOS2) gene in a noctuid pestSpodoptera litura.A fragment ofSlitBLOS2was identified by analyzing aS. lituratranscriptome database by local basic BLAST, and the full length cDNA was acquired by RACE strategy. To clarify the function ofSlitBLOS2, CRISPR/Cas9 based target mutagenesis ofSlitBLOS2was achieved, displaying a mosaic translucent integument in 62.3–70.6% larvae of G0 generation. Further PCR-based genotype analysis demonstrated various mutations occurred at theSlitBLOS2specific target site. A homozygote mutant individual was obtained in G1 generation, in which the yellow strips and white spots on the larval integument completely disappeared. Our study clearly demonstrates the function ofSlitBLOS2in the integument coloration, and thus provides a useful marker gene for genome editing based gene functional study and pest control strategy inS. lituraas well as other lepidopteran pests.