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

作　　者：Feng Wang Yan-Ting Ji Chi Tian Yuan-Cheng Wang Shen Xu Ri-Yuan Wang Qian-Qian Yang Ping Zhao Qing-You Xia 

机构地区：[1]State Key Laboratory of Silkworm Genome Biology,Chongqing Engineering and Technology Research Center for Novel Silk Materials,Biological Science Research Center,Southwest University,Chongqing,China

出　　处：《Insect Science》2021年第5期1277-1289,共13页昆虫科学（英文版）

基　　金：the National Natural Science Foundation of China(31530071);Chongqing Science and Technology C ommission(CSTC2018JCYJAX0298);Fundamental Research Funds for the Central Universities(XDJK2018C064);State Key Laboratory of Silkworm Genome Biology(SKLSGB1819-1).

摘　　要：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 phiC3I integrase-mediated promoter flip system(FLIP)for the inducible expression of target genes in silk-worm(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 phiC3 I-NLS integrase carrying a simian virus 40(SV40)nuclear localization signal(NLS)effectively flipped the BmAct4 promoter through an attBlattP 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 phiC3 I-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 offispring,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.

关 键 词：inducible gene expression PhiC31 integrase promoter flipping SILKWORM 

分 类 号：Q78[生物学—分子生物学]