An inducible constitutive expression system in Bombyx mori mediated by phiC31 integrase,Insect Science

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An inducible constitutive expression system in Bombyx mori mediated by phiC31 integrase
Insect Science ( IF 2.9 ) Pub Date : 2020-08-16 , DOI: 10.1111/1744-7917.12866
Feng Wang ₁ , Yan-Ting Ji ₁ , Chi Tian ₁ , Yuan-Cheng Wang ₁ , Shen Xu ₁ , Ri-Yuan Wang ₁ , Qian-Qian Yang ₁ , Ping Zhao ₁ , Qing-You Xia ₁

Affiliation

Inducible gene-expression systems play important roles in gene functional assays in the post-genome era. Streptomyces phage-derived phiC31 integrase, which mediates an irreversible site-specific cassette exchange between the phage attachment site (attP) and the bacterial attachment site (attB), provides a promising option for the construction of a controllable gene-expression system. Here, we report a phiC31 integrase-mediated promoter flip system (FLIP) for the inducible expression of target genes in silkworm (Bombyx mori). First, we constructed a FLIP reporter system, in which a BmAct4 promoter with enhanced translational efficiency was flanked by the attB and attP sites in a head-to-head orientation and further linked in a reverse orientation to a DsRed reporter gene. The coexpression of a C-terminal modified phiC31-NLS integrase carrying a simian virus 40 (SV40) nuclear localization signal (NLS) effectively flipped the BmAct4 promoter through an attB/attP exchange, thereby activating the downstream expression of DsRed in a silkworm embryo-derived cell line, BmE. Subsequently, the FLIP system, together with a system continuously expressing the phiC31-NLS integrase, was used to construct binary transgenic silkworm lines. Hybridization between FLIP and phiC31-NLS transgenic silkworm lines resulted in the successful flipping of the BmAct4 promoter, with an approximately 39% heritable transformation efficiency in silkworm offspring, leading to the constitutive and high-level expression of DsRed in silkworms, which accounted for approximately 0.81% of the silkworm pupal weight. Our successful development of the FLIP system offers an effective alternative for manipulating gene expression in silkworms and other lepidopteran species.

中文翻译：

phiC31整合酶介导的家蚕诱导型组成型表达系统

诱导型基因表达系统在后基因组时代的基因功能测定中发挥着重要作用。链霉菌噬菌体衍生的 phiC31 整合酶介导噬菌体附着位点 ( attP ) 和细菌附着位点 ( attB )之间不可逆的位点特异性盒交换，为构建可控基因表达系统提供了一个有前景的选择。在这里，我们报告了一种 phiC31 整合酶介导的启动子翻转系统 (FLIP)，用于在家蚕 (家蚕) 中诱导表达靶基因。首先，我们构建了一个 FLIP 报告系统，其中具有增强翻译效率的 BmAct4 启动子的两侧是attB和attP位点以头对头的方向连接，并进一步以反向连接到 DsRed 报告基因。携带猿猴病毒 40 (SV40) 核定位信号 (NLS) 的 C 端修饰的 phiC31-NLS 整合酶的共表达通过attB / attP交换有效翻转 BmAct4 启动子，从而激活DsRed的下游表达在蚕胚胎衍生的细胞系 BmE 中。随后，FLIP 系统与连续表达 phiC31-NLS 整合酶的系统一起用于构建二元转基因蚕系。FLIP 和 phiC31-NLS 转基因家蚕品系之间的杂交导致 BmAct4 启动子的成功翻转，家蚕后代的遗传转化效率约为 39%，导致家蚕中 DsRed 的组成型和高水平表达，约占蚕蛹重量的0.81%。我们成功开发的 FLIP 系统为操纵家蚕和其他鳞翅目物种的基因表达提供了一种有效的替代方法。

更新日期：2020-08-16

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