Elsevier

Gene

Volume 546, Issue 1, 1 August 2014, Pages 129-134
Gene

Identification of a functional element in the promoter of the silkworm (Bombyx mori) fat body-specific gene Bmlp3

https://doi.org/10.1016/j.gene.2014.05.038Get rights and content

Highlights

  • We characterized the promoter of the silkworm fat body-specific gene Bmlp3.

  • We identified a 21-bp core sequence upstream of the Bmlp3 transcription start site.

  • The 21-bp core sequence is essential for the transcriptional activity of the Bmlp3 promoter.

Abstract

30 K proteins are a group of structurally related proteins that play important roles in the life cycle of the silkworm Bombyx mori and are largely synthesized and regulated in a time-dependent manner in the fat body. Little is known about the upstream regulatory elements associated with the genes encoding these proteins. In the present study, the promoter of Bmlp3, a fat body-specific gene encoding a 30 K protein family member, was characterized by joining sequences containing the Bmlp3 promoter with various amounts of 5′ upstream sequences to a luciferase reporter gene. The results indicated that the sequences from − 150 to − 250 bp and − 597 to − 675 bp upstream of the Bmlp3 transcription start site were necessary for high levels of luciferase activity. Further analysis showed that a 21-bp sequence located between − 230 and − 250 was specifically recognized by nuclear factors from silkworm fat bodies and BmE cells, and could enhance luciferase reporter-gene expression 2.8-fold in BmE cells. This study provides new insights into the Bmlp3 promoter and contributes to the further clarification of the function and developmental regulation of Bmlp3.

Introduction

The fat body of the holometabolous insect Bombyx mori (silkworm) is a relatively large organ that is functionally similar to the vertebrate liver (Thomson, 1975). As a central storage center for nutrition and energy, the fat body synthesizes large amounts of proteins including lipoproteins, storage proteins and vitellogenins and secretes them into the hemolymph in a time-dependent manner during the life cycle of B. mori (Sakai et al., 1988, Wyatt and Pan, 1978). Among the major plasma proteins is a group of structurally related proteins with molecular weights around 30 kDa, termed B. mori low molecular lipoproteins (Bmlps or “30 K proteins”). 30 K proteins are synthesized largely in the fat body and play multiple roles in the growth and development of B. mori, including inhibiting apoptosis (Kim et al., 2001), defending against fungal infection (Ujita et al., 2005), translocating chymotrypsin inhibitor (Ueno et al., 2006), transporting lipid and/or sugar (Yang et al., 2011), improving sialylation of recombinant proteins (Wang et al., 2011), and enhancing cellular uptake and stability of enzymes (Lee et al., 2014). Interestingly, changes in the level of 30 K protein mRNA in the fat body reflect changes in the hemolymph concentration of 30 K proteins, indicating that transcription is the major mode of 30 K protein gene regulation. B. mori hemolymph proteins have proven to be good models for studying the developmental regulation of gene expression (Izumi et al., 1981, Mori et al., 1991a, Mori et al., 1991b).

The availability of the complete genome sequence of B. mori provides a unique opportunity to identify genes encoding 30 K proteins and determining how they are developmentally regulated. So far, a total of 24 genes encoding typical 30 K proteins have been identified (Bmlp1–24) (Zhang et al., 2012a, Zhang et al., 2012b). Most of the 30 K protein genes share similar genome organizations, as well as high similarity in their amino acid sequences (Mori et al., 1991a, Mori et al., 1991b, Sun et al., 2007). Notable, several genes such as Bmlp3 and Bmlp7, are synthesized largely in the fat body in a developmentally regulated manner (Deng et al., 2013, Zhang et al., 2012a, Zhang et al., 2012b). The biosynthesis of 30 K proteins appears to be repressed by juvenile hormone (JH) and appears to be the result of reduced transcription initiation (Bosquet et al., 1989, Izumi et al., 1984, Ogawa et al., 2005). These data suggest that there might be DNA elements responsive to JH in the upstream sequences of 30 K protein genes. Little is known about the promoters of 30 K protein genes and their associated enhancers.

In a previous study, we cloned a 1.1-kb promoter region of Bmlp3 and demonstrated its ability to direct fat body-specific expression of DsRed in transgenic silkworms (Deng et al., 2013). To further characterize the promoter of Bmlp3, a series of promoter-containing DNA fragments of differing lengths were attached to a luciferase reporter gene and the levels of reporter-gene expression were measured. Our results indicate the presence of a 21-bp sequence located between − 230 and − 250 bp upstream of the Bmlp3 transcription start site with the ability to increase luciferase gene expression in BmE cells.

Section snippets

Cell culture and chemicals

The B. mori embryonic cell line BmE and the Spodoptera litura embryonic cell line Spli-221, were maintained at 27 °C in Grace's medium plus 10% fetal bovine serum (FBS, Hyclone, China), supplemented with 50 U/mL penicillin and 50 mg/mL streptomycin. The insect hormones, juvenile hormone III (JH III) and 20-hydroxyecdysone (20E) (Sigma, USA), were used to evaluate the effects of hormones on promoter activity.

Generation of the Bmlp3 promoter deletion constructs

Bmlp3 promoter constructs containing varying amounts of 5′ upstream sequences were

Effects of insect hormones on the Bmlp3 promoter activity

In silkworms, hormones and especially 20E and JH mainly function as regulators of ecdysis and metamorphosis (Soin et al., 2008, Tang et al., 2003). Previous studies indicated that there were putative ecdysone- and JH-response elements within 1.1-kb of the Bmlp3 promoter, and that the biosynthesis of 30 K proteins could be repressed directly or indirectly by JH (Deng et al., 2013, Izumi et al., 1984, Ogawa et al., 2005). Therefore, we investigated further whether insect hormones were involved in

Conclusions

In this study, the promoter of a fat body-specific gene encoding a member of the 30 K protein family, Bmlp3, was characterized by creating various luciferase reporter gene constructs with differing amounts of 5′ promoter-proximal sequences. A 21-bp core sequence located between − 230 and − 250 upstream of transcription start site of Bmlp3 was identified using the EMSA method. We demonstrated that the 21-bp sequence can bind specifically with the nuclear proteins from the fat body of B. mori and

Conflict of interest

The authors declare no conflict of interest.

Acknowledgments

We thank Professor Qili Feng of South China Normal University, China, for providing the Spodoptera litura embryonic cell line Spli-221. We are grateful to Professor David O'Brochta of the University of Maryland, USA, and Huawei He of Southwest University, China, for improving this manuscript. This work was supported by the National Basic Research Program of China (2012CB114600), and Grant (XDJK2014B014) from the Fundamental Research Funds for the Central Universities.

References (22)

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