Effects of Certain cis-Regulatory Elements on Stage-Specific vitellogenin Expression in the Bombyx mori (Lepidoptera: Bombycidae)

Abstract

Bombyx mori vitellogenin (BmVg) is highly upregulated during pupation, and the 20-hydroxyecdysone and amino acids may regulate stage-specific BmVg expression. However, previous studies showed that other factors may also affect stage-specific BmVg expression. Here, we characterized effective BmVg transcription factors by identifying the corresponding cis-regulatory elements (CREs). We prepared transgenic B. mori, in which DsRed was driven by various lengths of BmVg promoter. qRT-PCR analysis showed that DsRed expression driven by a 1.0-kb BmVg promoter (VgP1.0K) was consistent with endogenous BmVg. VgP1.0K specificity was closer to the endogenous BmVg promoter than that of VgP0.8K. These results suggest that CREs affecting stage-specific BmVg expression were localized to the 1.0-kb BmVg promoter. We investigated the effects of certain CREs that could influence the stage specificity of BmVg promoter on BmVg expression in transgenic B. mori. The relative DsRed expression was significantly reduced in transgenic female B. mori and the peak in DsRed expression was delayed after E-box CRE mutation. These results demonstrate that the E-box element enhanced BmVg expression and also affected stage-specific BmVg expression. Moreover, the relative DsRed expression was significantly increased in transgenic female of B. mori after 3×BD CRE mutation in BmVg promoter. However, the stage specificity of the mutated promoter was consistent with that of the endogenous BmVg promoter. The 3×BD element downregulated BmVg but had no effect on stage-specific BmVg expression. The present study promoted the process of elucidating the regulatory network for stage-specific BmVg expression and furnished a theoretical basis for the application of BmVg promoter.

Keywords: B. mori; promoter; regulation; transgenic; vitellogenin.

Fig. 1.

Generation of transgenic B. mori with DsRed driven by the BmVg promoter. (A) Activity of BmVg promoters with different lengths. (B) Map of transgenic vector; pBacR and pBacL are the right and left arms of the transposon, respectively; 3xp3 EGFP SV40 is the transgene marker; and Promoter is the BmVg promoter. (C) Screening of positive transgenic B. mori under fluorescence microscope; (a1–d1) white light image of wild-type B. mori (a1), VgP0.8K transgenic B. mori (b1), VgP1.0K transgenic B. mori (c1), and VgP1.7K transgenic B. mori (d1); (a2–d2) fluorescence image of wild-type B. mori (a2), VgP0.8K transgenic B. mori (b2), VgP1.0K transgenic B. mori (c2), and VgP1.7K transgenic B. mori (d2). (D) Analysis of insertion sites for transgenic B. mori. Significant differences between data sets were calculated by two-tailed Student’s t-tests; *P < 0.05; **P < 0.01.

Fig. 2.

Detection of exogenous DsRed expression in various B. mori by RT-PCR. (A–D) DsRed expression in fat bodies at different B. mori stages. (A) Wild-type B. mori. (B) BmVgP0.8K transgenic B. mori. (C) BmVgP1.0K transgenic B. mori. (D) BmVgP1.7K transgenic B. mori. (E) DsRed expression in various B. mori tissues. BmVg was a positive control and BmActin3 was a housekeeping gene. (W1–W3) Wandering stage (d); (P1, P3) pupal stage (d).

Fig. 3.

Detection of exogenous DsRed expression in various transgenic B. mori. (A) DsRed expression trend in BmVgP0.8K transgenic B. mori. (B) DsRed expression trend in BmVgP1.0K transgenic B. mori. (C) DsRed expression trend in BmVgP1.7K transgenic B. mori. (D) Relative expression of DsRed in various transgenic B. mori. Significant differences between data sets calculated by two-tailed Student’s t-tests; **P < 0.01.

Fig. 4.

Effect of E-box on BmVg promoter activity. (A) Position of E-box element on BmVg promoter. (B) Effect of E-box element on BmVg promoter activity in BmE-SWU1 cell. (C) Activity of 78ML basal promoter linked with E-box CRE. (D) Effects of E-box CRE on BmVg promoter activity after mutation. (E) qPCR of DsRed expression trend in BmVgP1.0M transgenic B. mori. (F) Comparison of DsRed expression on day 3 after wandering in female of transgenic B. mori. For more visual representation relative fold change in promoter activity after mutation, the DsRed expression driven by BmVgP1.0K promoter without mutation was normalized as 1. Significant differences between data sets calculated by two-tailed Student’s t-tests; *P < 0.05; **P < 0.01.

Fig. 5.

Effects of 3×BD CRE on BmVg promoter activity. (A) Position of 3×BD CRE in BmVg promoter. (B) Conservative core motif of DREF and BEAF CRE. (C) Effect of 3×BD CRE on BmVg promoter activity in BmE-SWU1 cell. (D) qPCR of DsRed expression trend in BmVgP1.7K-3×BD transgenic B. mori. (E) Comparison of DsRed expression on day 3 after wandering in female transgenic B. mori. DsRed expression in W3 of BmVgP1.7K female transgenic B. mori was normalized as 1. Significant difference between data sets calculated by two-tailed Student’s t-tests; **P < 0.01.