Functional characterization of the vitellogenin promoter in the silkworm, Bombyx mori

Abstract

Genetic transformation and genome editing technologies have been successfully established in the lepidopteran insect model, the domesticated silkworm, Bombyx mori, providing great potential for functional genomics and practical applications. However, the current lack of cis-regulatory elements in B. mori gene manipulation research limits further exploitation in functional gene analysis. In the present study, we characterized a B. mori endogenous promoter, Bmvgp, which is a 798-bp DNA sequence adjacent to the 5'-end of the vitellogenin gene (Bmvg). PiggyBac-based transgenic analysis shows that Bmvgp precisely directs expression of a reporter gene, enhanced green fluorescent protein (EGFP), in a sex-, tissue- and stage-specific manner. In transgenic animals, EGFP expression can be detected in the female fat body from larval-pupal ecdysis to the following pupal and adult stage. Furthermore, in vitro and in vivo experiments revealed that EGFP expression can be activated by 20-hydroxyecdysone, which is consistent with endogenous Bmvg expression. These data indicate that Bmvgp is an effective endogenous cis-regulatory element in B. mori.

Functional characterization of the cis‐regulatory region for the vitellogenin gene in Plutella xylostella

“…The 510 and 200 bp genomic sequences with TTAA duplication insertion sites of the vector were identified at the 5 0 and 3 0 ends, respectively. (Xu et al, 2014;Yano et al, 1994), which is similar to our PxVgP. We also identified two binding sites of BrC-Z2 in PxVgP2; these BrC-Z2s play roles as ecdysone steroid response elements in Drosophila and Ae.…”

“…In B. mori , the Bmdsx F isomers bind at dsx F binding sites in the transcription initiation site of the BmVg gene and regulate the expression of the Vg gene in females (Suzuki et al ., ). These Bmdsx F binding sites were identified in the B. mori Vg promoter (Xu et al ., ; Yano et al ., ), which is similar to our PxVgP . We also identified two binding sites of BrC ‐ Z2 in PxVgP2 ; these BrC ‐ Z2 s play roles as ecdysone steroid response elements in Drosophila and Ae.…”

“…stephensi (Nirmala et al ., ), An. gambiae (Chen et al ., ) and B. mori (Xu et al ., ), VgP is a late acting promoter and promotes gene expression in later stages in specific tissues. Low activity in the embryonic cell line might be a result of the tissue‐specific and late acting nature of this promoter, and it is necessary to determine the activity in the fat‐body cell line.…”

“…The core part of the promoter is a short DNA sequence that contains an RNA polymerase II binding site and some other general transcriptional factors such as TFIIA, TFIIB, TFIID, TFIIE, TFIIF and TFIIH, and is located upstream of the transcription start site of the gene (Ye et al ., ). The germline‐, tissue‐, sex‐ and stage‐specific cis ‐regulatory promoter elements have been extensively studied in lepidopteran insects, such as the female‐fat‐body‐specific promoter (Xu et al ., ), eye‐specific promoter (Thomas et al ., ), silk‐gland‐specific promoter (Tomita et al ., ), testis‐specific promoter (Xu et al ., ), nanos promoter (Xu et al ., ) and actin3 promoter (Fatyol et al ., ). The demand for alternative cis ‐regulatory elements is going to increase because of the development of transgenic insects for pest management, and it is necessary to identify more cis ‐regulatory elements (such as stage‐, tissue‐ and sex‐specific ones) for precise and efficient gene manipulation in lepidopteran insects.…”

“…Vitellogenin (Vg) is a precursor of yolk protein and mostly synthesized in the fat body (Raikhel, ; Tufail and Takeda, ; Upadhyay et al ., ; Veerana et al ., ). The sex‐, stage‐ and tissue‐specific expression of the Vg gene and Vg cis‐regulatory elements have been extensively studied in Bombyx mori (Xu et al ., ), Anopheles stephensi (Nirmala et al ., ), Aedes aegypti (Kokoza and Raikhel, ) and Anopheles gambiae (Chen et al ., ).…”