Identification of a germline-expression promoter for genome editing in Bombyx mori
Identification of a germline-expression promoter for genome editing in Bombyx mori
复制标题
鉴定用于家蚕基因组编辑的种系表达启动子
DOI：
10.1111/1744-7917.12657
复制
发表时间：
2019-12-01
期刊：
影响因子：
4
通讯作者：
Huang, Yong-Ping
中科院分区：
文献类型：
--
作者：
Xu, Jun;Chen, Rong-Mei;Huang, Yong-Ping
阶段和组织特异性顺式调控元件的鉴定将使更精确的基因组编辑成为可能。在以前的研究家蚕，我们确定了几个组织和性别特异性的顺式调控元件，利用转基因技术，包括女性和脂肪体特异性启动子，卵黄蛋白原，睾丸特异性启动子，辐射辐头1（BmR 1）和β-微管蛋白4（Bm β 4）。在这里，我们报告一个顺式调控元件体细胞和生殖细胞表达的启动子，纳米（Bmnos）。我们研究了Bmnos转录起始位点上游三个截短的启动子序列（nos-0.6kb、nos-1 kb和nos-2kb）在体外和体内的活性。在BmN培养的细胞中，所有三种长度驱动编码增强型绿色荧光蛋白（EGFP）的基因的表达，尽管nos-2kb具有最高的荧光活性。在转基因家蚕中，nos-2kb在胚胎早期驱动EGFP表达，并且在胚胎后期荧光集中在性腺中。此外，该顺式调节元件没有性别分化。荧光强度随幼虫的发育而逐渐减弱，在生殖腺中广泛表达。nos-2kb启动子以与广谱强IEl启动子相当的效率驱动Cas9系统。这些结果表明，Bmnos是早期胚胎和性腺中有效的内源性顺式调节元件，其可用于涉及成簇的、规则间隔的短回文重复序列（CRISPR）/Cas9系统的应用中。
Identification of stage- and tissue-specific cis-regulatory elements will enable more precise genomic editing. In previous studies of the silkworm Bombyx mori, we identified and characterized several tissue- and sex-specific cis-regulatory elements using transgenic technology, including a female- and fat body-specific promoter, vitellogenin, testis-specific promoters, Radial spoke head 1 (BmR1) and beta-tubulin 4 (Bm beta 4). Here we report a cis-regulatory element specific for a somatic and germ cell-expressed promoter, nanos (Bmnos). We investigated activities of three truncated promoter sequences upstream of the transcriptional initiation site sequences of Bmnos in vitro (nos-0.6kb, nos-1kb and nos-2kb) and in vivo (nos-2kb). In BmN cultured cells, all three lengths drove expression of the gene encoding enhanced green fluorescence protein (EGFP), although nos-2kb had the highest fluorescence activity. In transgenic silkworms, nos-2kb drove EGFP expression at the early embryonic stage, and fluorescence was concentrated in the gonads at later embryonic stages. In addition, this cis-regulatory element was not sex differentiated. The fluorescence intensity gradually weakened following the larval developmental stage in the gonads and were broadly expressed in the whole body. The nos-2kb promoter drove the Cas9 system with efficiency comparable to that of the broad-spectrum strong IE1 promoter. These results indicate that Bmnos is an effective endogenous cis-regulatory element in the early embryo and in the gonad that can be used in applications involving the clustered, regularly interspaced, short palindromic repeats (CRISPR)/Cas9 system.