Screening and optimization of an efficient Bombyx mori nucleopolyhedrovirus inducible promoter

Cao, Mengji; Kuang, Xiu-Xiu; Li, Haiqing; Lei, Xue-jiao; Xiao, Wen‐Fu; Dong, Zhanqi; Zhang, Jun; Hu, Nan; Chen, Tingting; Lü, Cheng; Pan, Min‐Hui

doi:10.1016/j.jbiotec.2016.05.037

Cited by 11 publications

(13 citation statements)

References 39 publications

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“…In addition, insects can also use RNAi, an innate immunity against viruses, where double‐stranded RNA encoded by the virus acts as the pathogen trigger to resist viral invasion (Wang et al, ). Previous studies show that interfering with the replication of necessary genes, including the ie‐1 , ie‐2 , lef‐1 and gp64 genes of B. mori nuclearpolyhedrosis virus (BmNPV), through RNAi can effectively inhibit BmNPV proliferation (Zhang et al, ; Cao et al, ). Jiang & Xia () demonstrated that RNAi of the ie‐2 gene significantly inhibits BmNPV proliferation.…”

Section: Ampsmentioning

confidence: 99%

“…et al, 2006). Previous studies show that interfering with the replication of necessary genes, including the ie-1, ie-2, lef-1 and gp64 genes of B. mori nuclearpolyhedrosis virus (BmNPV), through RNAi can effectively inhibit BmNPV proliferation Cao et al, 2016). Jiang & Xia (2013) demonstrated that RNAi of the ie-2 gene significantly inhibits BmNPV proliferation.…”

Section: Other Routes Of the Immune Responsementioning

confidence: 99%

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Research progress on the immune mechanism of the silkworm Bombyx mori

et al. 2018

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The silkworm Bombyx mori L., representing an important economic insect and one of the best models for studying insect immunity, possesses an efficient and sophisticated innate immune system against invasive microorganisms. The innate immune system basically includes humoural immunity and cellular immunity. The humoural immunity, which functions via molecules including humoural factors, lysozymes, phenoloxidase, hemolin, lectins and, in particular, antimicrobial peptides, plays a central role in eliminating the invading pathogens. The cellular immunity is primarily carried out and mediated by plasmatocytes and granular cells of haemocytes in the haemolymph, usually followed by melanization. Additionally, apoptosis, a primary viral defence for insects lacking adaptive immunity, comprises an important part of the silkworm immune system. Currently, there is still the lack of a comprehensive and systematic understanding of the molecular mechanisms of silkworm immunity. We review the latest research progress on silkworm immune mechanisms, including phenoloxidase‐dependent melanization and apoptosis, which is conducive to improving our understanding of the silkworm immune mechanism, clarifying the relationship of various immune mechanisms, and also providing a theoretical basis and reference for the future research of insect immunity.

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Section: Ampsmentioning

confidence: 99%

Section: Other Routes Of the Immune Responsementioning

confidence: 99%

Research progress on the immune mechanism of the silkworm Bombyx mori

et al. 2018

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“…Previous studies have shown that the -773 bp upstream and +134 bp downstream motifs of the 39K promoter transcription initiation site are critical regions for 39K promoter activity (19). To analyze the structural features of the 39K promoter, we performed a stepwise truncation analysis of the 39K promoter.…”

Section: Methodsmentioning

confidence: 99%

“…We previously screened for the B. mori nucleopolyhedrovirus (BmNPV)-induced promoter (VP1054, P33, Bm21, Bm122, 39K, P143 and P6.9) activity and found that the 39K promoter had the highest BmNPV-induced transcriptional activity (19). The virus-inducing activity of the BmNPV 39K promoter could be further increased using enhancers such asHr3, Hr5, Polh and PU (19). Simultaneously, the overexpression of an exogenous hycu-ep32 gene controlled by an inducible 39K promoter shows high antiviral capacity in transgenic lines (20).…”

Section: Introductionmentioning

confidence: 99%

Construction and Characterization of a Synthetic Baculovirus-inducible 39K Promoter

Dong

et al. 2018

Preprint

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The low expression activity and specificity of natural promoters limit the 22 applications of genetic engineering. To construct a highly efficient synthetic inducible 23 promoter in the Bombyx mori (Lepidoptera), we analyzed the regulatory elements and 24 functional regions of the B. mori nucleopolyhedrovirus (BmNPV) 39K promoter. The 25 results of truncated mutation analysis of the 39K promoter showed that the 26 transcriptional regulatory region spanning positions -573 to -274 and +1 to +62 is 27 essential for virus-inducible promoter activity. Further investigation using 28 electrophoretic mobility shift assay (EMSA) revealed that the baculovirus IE-1 29 protein binds to the 39K promoter at the -310 to -355 region, and transcription 30 activates the expression of 39K promoter assay. Finally, we successfully constructed a 31 synthetic inducible promoter that increase the virus-inducing activity of other 32 promoters using the baculovirus-inducible transcriptional activation region that binds 33 to specific core elements of 39K (i.e., spanning the region -310 to -355) . In summary, 34 we describes a novel, synthetic, and highly efficient biological tool, namely, a 35 virus-inducible 39K promoter, which provides endless possibilities for future gene 36 function research, gene therapy, and pest control in genetic engineering.37 38

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“…The majority of previous antiviral studies have reported the use of knockdown of baculovirus DNA replication factor immediate early‐1 (IE‐1) gene through RNAi technology, which significantly inhibited the virus replication in vivo and in vitro (Zhang, Wang et al , ; Zhou et al , ). Moreover, ie‐1 , lef‐1 , lef‐2 , lef‐3 , lef‐11 , oral infection factor p74 and gp64 have also significantly inhibited virus multiplication and replication in transgenic cells and silkworm (Fujita et al , ; Jiang et al , ; Xu et al , ; Zhang, He et al , ; Zhou et al , ; Cao et al , ). However, baculovirus early transcription activator and DNA replication factors, ie‐0 and ie‐2 , have not yet been studied as target genes.…”

Section: Introductionmentioning

confidence: 99%

CRISPR/Cas9‐mediated disruption of the immediate early‐0 and 2 as a therapeutic approach to Bombyx mori nucleopolyhedrovirus in transgenic silkworm

Dong

et al. 2018

Insect Molecular Biology

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The CRISPR/Cas9 system is a powerful tool for the treatment of infectious diseases. In our previous study, we knocked out the Bombyx mori nucleopolyhedrovirus (BmNPV) key genes and BmNPV-dependent host factor to generate transgenic antiviral strains. To further expand the range of target genes for BmNPV and more effectively prevent and control pathogenic infections, we performed gene editing and antiviral analysis by constructing a target-directed baculovirus early transcriptional activator immediate early-0 (ie-0) and 2 (ie-2) transgenic silkworm line. We hybridized it with Cas9 transgenic line to produce a double-positive transgenic Cas9(+)/sgIE0-sgIE2(+) line that could activate the CRISPR gene editing system. We first demonstrated that the system is capable of efficiently editing target genes and resulting in fragment deletions in the BmNPV genome. Survival rate of the transgenic Cas9(+)/sgIE0-sgIE2(+) line reached 65% after inoculation with 1 × 10 occlusion bodies/larva. Molecular analysis showed that BmNPV DNA replication and viral gene expression level in the transgenic Cas9(+)/sgIE0-sgIE2(+) line were significantly inhibited compared with the control Cas9(-)/sgIE0-sgIE2(-) line. These results indicated that IE-0 and IE-2, as baculovirus early transcriptional activators, can be used as target sites for gene therapy and that multigene editing could expand the range of target sites for research to create silkworm resistance breeds.

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Screening and optimization of an efficient Bombyx mori nucleopolyhedrovirus inducible promoter

Cited by 11 publications

References 39 publications

Research progress on the immune mechanism of the silkworm Bombyx mori

Research progress on the immune mechanism of the silkworm Bombyx mori

Construction and Characterization of a Synthetic Baculovirus-inducible 39K Promoter

CRISPR/Cas9‐mediated disruption of the immediate early‐0 and 2 as a therapeutic approach to Bombyx mori nucleopolyhedrovirus in transgenic silkworm

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