The intronic promoter of Actin4 mediates high-level transgene expression mainly in the wing and epidermis of silkworms

Tan, Tingting; Liu, Rongpeng; Luo, Qiong; Ma, Jingwen; Ou, Yao; Zeng, Wenhui; Feng, Lichun; Xu, Hanfu

doi:10.1007/s11248-020-00192-0

Cited by 3 publications

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“…pGL4.11[ Luc2P ] was used as a negative control. The plasmids p57S[hrA4- Gal4 ] (A4G4) and pGL4.11[UAS- Luc2P ] (UAS-Luc2P), which we constructed based on a previously modified Gal4/UAS binary system 23 , 24 , were used as positive controls.…”

Section: Methodsmentioning

confidence: 99%

New insights into the proteins interacting with the promoters of silkworm fibroin genes

Luo

et al. 2021

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The silkworm, Bombyx mori, is a silk-producing insect that has contributed greatly to human society. The silk gland of B. mori is a specialized organ responsible for synthesizing silk fibroin and sericin proteins under control of numerous factors. However, which factors are involved in direct silk protein synthesis regulation remains largely unknown. We report the identification of promoter-interacting proteins (PIPs) necessary for the regulation of genes encoding fibroin proteins, including the fibroin heavy chain (fibH), fibroin light chain (fibL), and a 25-kD polypeptide protein (P25). In the fourth larval molting stage (M4) or day 5 fifth-instar larvae (L5D5), a total of 198, 292, and 247 or 330, 305, and 460 proteins interacting with the promoter region of fibH, fibL and P25, respectively, were identified from the posterior silk gland by DNA pull-down combined with mass spectrometry. Many PIPs were particularly involved in ribosome- and metabolism-related pathways. Additionally, 135 and 212 proteins were identified as common PIPs of fibH, fibL and P25 in M4 and L5D5, respectively. Among all PIPs, we identified 31 potential transcription factors, such as Y-box and poly A-binding proteins, which play roles in nucleotide binding, ATP binding, or protein folding. This study provides the first in-depth profile of proteins interacting with fibroin gene promoters and contributes to a better understanding of silk protein synthesis regulation.

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Section: Methodsmentioning

confidence: 99%

New insights into the proteins interacting with the promoters of silkworm fibroin genes

Luo

et al. 2021

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“…11[Luc2P] was used as a negative control. The plasmids p57S[hrA4-Gal4] (A4G4) and pGL4.11[UAS-Luc2P] (UAS-Luc2P), which we constructed based on a previously modi ed Gal4/UAS binary system 23,24 , were used as positive controls.…”

Section: Methodsmentioning

confidence: 99%

New insights into the proteins interacting with the promoters of silkworm fibroin genes

Luo

et al. 2021

Preprint

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The silkworm, Bombyx mori, is a silk-producing insect that has contributed greatly to human society. The silk gland of B. mori is a specialized organ responsible for synthesizing silk fibroin and sericin proteins under control of numerous factors. However, which factors are involved in direct silk protein synthesis regulation remains largely unknown. We report the identification of promoter-interacting proteins (PIPs) necessary for the direct regulation of genes encoding fibroin proteins, including the fibroin heavy chain (fibH), fibroin light chain (fibL), and a 25-kD polypeptide protein (P25). In the fourth larval molting stage (M4) or day 5 fifth-instar larvae (L5D5), a total of 198, 292, and 247 or 330, 305, and 460 proteins binding to fibH, fibL and P25, respectively, were identified from the posterior silk gland by DNA pull-down combined with mass spectrometry. Many PIPs were particularly involved in ribosome-, metabolism- and disease-related pathways. Additionally, 135 and 212 proteins were identified as common PIPs of fibH, fibL and P25 in M4 and L5D5, respectively. Among all PIPs, we identified 31 potential transcription factors, such as Y-box and poly A-binding proteins, which play roles in nucleotide binding, ATP binding, or protein folding. This study provides the first in-depth profile of proteins interacting with fibroin gene promoters and contributes to a better understanding of silk protein synthesis regulation.

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“…Recently, we established a transgenic silkworm line (named A4G4) that expresses the Gal4 mainly in the wing disc (WD) and epidermis (EP) under the control of the promoter of the B. mori Actin4 gene [21]. This line is a good material for evaluation of the effects of Gal4 protein expression on normal gene expression in transgenic tissues.…”

Section: Introductionmentioning

confidence: 99%

Transcriptome analysis reveals the effects of transgenic expression of the Gal4 protein on normal gene expression in silkworm tissues

et al. 2020

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25The Gal4/upstream activating sequence(UAS) system, a well-known genetic tool, has been widely 26 used to analyze gene function in many organisms, including the silkworm (Bombyx mori), a model 27 lepidopteran insect. Several studies have suggested that Gal4 protein activation in tissues can 28 negatively affect transgenic individuals; however, whether and to what extent the Gal4 protein 29 affects normal endogenous gene expression have rarely been studied. Here, we analyzed the 30 transcriptomes of transgenic silkworms expressing the Gal4 protein at high levels in both the wing 31 disc (WD) and epidermis (EP) and investigated gene expression changes in both tissues. Overall, 32 24,593 genes were identified in the WD and EP libraries, and 2,025 and 2,488 were identified as 33 significant differentially expressed genes(DEGs) in the WD and EP between the transgenic and 34 control groups, respectively. These DEGs were further annotated by gene function classification and 35 pathway assessment using public databases. In addition, 506 DEGs were shared (common) between 36 both tissues. Of these, 97 genes were commonly upregulated, and 234 were commonly 37 downregulated; many of them were annotated to be involved in metabolic processes such as "fat 38 digestion and absorption", "glycine, serine and threonine metabolism" and "glutathione metabolism" 39 and in signal transduction pathways such as the "Rap1 signaling pathway", "MAPK signaling 40 pathway" and "Hippo signaling pathway". Overall, this work enhances understanding of the effects 41 of transgenic Gal4 protein expression on normal gene expression in silkworm tissues and suggests 42 that researchers should pay attention to unexpected effects when using the Gal4/UAS system to study 43 gene function. 44 Introduction 46The Gal4/upstream activating sequence(UAS) binary expression system, derived from yeast and 47 originally developed in Drosophila [1-3], is a powerful genetic tool that allows manipulation of 48 target gene expression in a spatiotemporally precise fashion. Since its first application in Drosophila, 49 the Gal4/UAS system has been widely used to analyze gene function in dozens of organisms, 50 including mice [4], zebrafish [5], Xenopus [6], Bombyx mori [7], Arabidopsis thaliana [8], 51 Triboliumcastaneum [9], Aedes aegypti [10], Anopheles stephensi [11] and Caenorhabditis elegans 52[12]. The Gal4/UAS system has also been employed to develop novel genetic tools, such as the 53 enhancer/gene trap system and the Q system [13][14][15], and it has been combined with genome editing 54 tools for conditional manipulation of gene expression in vivo [16][17]. 55 In recent decades, remarkable progress in gene function analysis has been achieved with the 56 Gal4/UAS system. However, the fact that high protein levels of Gal4 have certain toxicity toward 57 cells coexpressing UAS-linked target genes and Gal4 protein cannot be ignored. Although some 58 researchers have described and developed novel Gal4/UAS systems with smaller sizes but greater 59 transactivati...

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The intronic promoter of Actin4 mediates high-level transgene expression mainly in the wing and epidermis of silkworms

Cited by 3 publications

References 23 publications

New insights into the proteins interacting with the promoters of silkworm fibroin genes

New insights into the proteins interacting with the promoters of silkworm fibroin genes

New insights into the proteins interacting with the promoters of silkworm fibroin genes

Transcriptome analysis reveals the effects of transgenic expression of the Gal4 protein on normal gene expression in silkworm tissues

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