家蚕β-呋喃果糖苷酶基因BmSuc1的 CRISPR敲除载体的构建与导入
Construction of a CRISPR vector to edit the β-fructofuranosidase gene BmSuc1 and its incorporation in the silkworm Bombyx mori
朱文恺1** 甘 泉1, 2** 贺 伟1 张新伟1, 2 周 跃1 吴梦雪1 孙同
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DOI：10.7679/j.issn.2095-1353.2018.029
作者单位：(1. 安徽农业大学生命科学学院，合肥 230036；2.安徽省蚕桑资源利用国际联合研发中心，合肥 230036
中文关键词： CRISPR/Cas9，基因编辑，sgRNA，家蚕，β-呋喃果糖苷酶
英文关键词：CRISPR/Cas9, genome edition, sgRNA, Bombyx mori, β-fructofuranosidase
中文摘要：
【目的】 CRISPR/Cas9是近些年报道较多的基因编辑新工具，具有简便高效、特异性强等优势。BmSuc1是从鳞翅目经济昆虫家蚕Bombyx mori体内发现的编码β-呋喃果糖苷酶（β-fructofuranosidase，β-FFase）的基因，也是首个被克隆和鉴定的动物型β-FFase编码基因。β-FFase是作用于果糖基的蔗糖水解酶，BmSUC1可能与家蚕防御桑叶生物碱的生理过程有关，但目前其发挥作用的分子机制尚不清晰。为了解析BmSuc1在蚕体的作用途径及其生理功能，本研究基于CRISPR/Cas9基因编辑系统构建表达双元sgRNA的CRISPR载体，用于敲除家蚕基因组中的BmSuc1基因。【方法】 根据目的基因BmSuc1的ORF序列设计2条sgRNA，通过同源重组的方法分别插入CRISPR载体的Sal I和Nhe I酶切位点。进而利用转基因显微注射技术将该编辑载体注入G0代蚕卵，经催青孵化饲养家蚕并自交制备G1代蚕种。【结果】 PCR验证及测序结果均表明CRISPR编辑载体构建成功。根据DsRed2红色蛋白的荧光标记，从G1代家蚕幼虫中成功筛选出阳性转基因个体。【结论】 本文详细介绍了表达双元sgRNA的CRISPR载体的构建方法，所获得的阳性转基因家蚕对于下一步探讨BmSuc1在家蚕糖类营养的吸收与利用途径中的作用奠定了重要的实验基础，有助于阐明蚕-桑相互选择和适应的分子机制。
英文摘要：
[Objectives] CRISPR/Cas9 is a novel genome-editing tool that has recently had broad application in many fields of research. CRISPR/Cas9 has the advantages of simplicity, efficiency and high specificity. BmSuc1 encodes β-fructofuranosidase (β-FFase, a fructosyl-hydrolytic sucrase) in the silkworm Bombyx mori (Lepidoptera). BmSuc1 is also the first β-FFase gene to have been cloned and identified in an animal. The physiological function of BmSUC1 in the silkworm is probably related to defense against mulberry alkaloids. However, further research is needed to confirm this. In order to clarify the role of BmSUC1 we used the CRISPR/Cas9 genome-editing system to construct a dual-sgRNA expressing vector. [Methods] We first designed two sgRNAs against BmSuc1 ORF and inserted these into the Sal I and Nhe I restriction enzyme sites of a CRISPER edition vector using the homologous recombination method. We then microinjected the recombinant CRISPR plasmid into G0 silkworm eggs and created a G1 generation by G0 sib-crossing. [Results] Both sgRNA insertions were verified by polymerase chain reaction and sequencing. DsRed2 fluorescence confirmed that we successfully obtained positive transgenic G1 individuals. [Conclusion] This study provides a detailed description of the construction of a CRISPR edition vector to express dual sgRNAs. The resultant positive transgenic silkworms will help clarify the biological role of BmSuc1-coding β-FFase in the silkworm.