Transgenic characterization of two testis‐specific promoters in the silkworm, Bombyx mori

Abstract

Sex-specific regulatory elements are key components for developing insect genetic sexing systems. The current insect genetic sexing system mainly uses a female-specific modification system whereas little success was reported on male-specific genetic modification. In the silkworm Bombyx mori, a lepidopteran model insect with economic importance, a transgene-based, female-specific lethality system has been established based on sex-specific alternative splicing factors and a female-specific promoter BmVgp (vitellogenin promoter) has been identified. However, no male-specific regulatory elements have yet been identified. Here we report the transgenic identification of two promoters that drive reporter gene expression in a testis-specific manner in B. mori. Putative promoter sequences from the B. mori Radial spoke head 1 gene (BmR1) and beta-tubulin 4 gene (Bmβ4) were introduced using piggybac-based germline transformation. In transgenic silkworms, expression of the reporter gene enhanced green fluorescent protein (EGFP) directed by either BmR1 promoter (BmR1p) or Bmβ4p showed precisely testis-specific manners from the larval to adult stage. Furthermore, EGFP expression of these two transgenic lines showed different localization in the testis, indicating that BmR1p or Bmβ4p might be used as distinct regulatory elements in directing testis-specific gene expression. Identification of these testis-specific promoters not only contributes to a better understanding of testis-specific gene function in insects, but also has potential applications in sterile insect techniques for pest management.

Transgenic Expression of the piRNA-Resistant Masculinizer Gene Induces Female-Specific Lethality and Partial Female-to-Male Sex Reversal in the Silkworm, Bombyx mori

“…RT-PCR detected an approximately 850 bp of BmRI cDNA from +/+ and Masc-R /+ testes but not from +/+ ovary (Fig 5J, compare lane 1 with lanes 3 and 4). Thus, our RT-PCR analysis confirmed the testis-specific expression of BmR1 as reported previously [32]. The RT-PCR analysis also showed that BmR1 was expressed not only in testes, but also in the Masc-R /+ ovaries of the fifth instar larvae (Fig 5J, lanes 2 and 4).…”

“…To examine whether the Masc-R /+ ovaries contain tissues partially developed into testis, we performed RT-PCR analysis using primers that anneal to the B . mori homolog of radial spoke head protein 1 gene ( BmR1 ), which is specifically expressed in the testis [32]. RT-PCR detected an approximately 850 bp of BmRI cDNA from +/+ and Masc-R /+ testes but not from +/+ ovary (Fig 5J, compare lane 1 with lanes 3 and 4).…”

Transgenic characterization of two silkworm tissue‐specific promoters in the haemocyte plasmatocyte cells

“…Xu et al . () investigated the promoter activities of Radial spoke head 1 and beta‐tubulin 4 with transgenic technology and found that the promoters showed only testis‐specific activity. In our laboratory, promoters specific to the midgut (Jiang et al ., ) and to the fat body (Deng et al ., ) were also successfully identified using the transgenic system.…”

“…Experimental results show that these two promoters have great potential for the mass production of recombinant proteins of biomedical and pharmaceutical interest in silkworms after transformation (Tatematsu et al ., ). In addition, tissue‐specific promoters also have been reported in the midgut and testes (Jiang et al ., ; Xu et al ., ).…”

Highly efficient and inducible DNA excision in transgenic silkworms using the FLP/FRT site-specific recombination system

“…Actually, other tissue-specific promoters, such as silk gland- (Tomita et al 2003(Tomita et al , 2007Long et al 2015b), fat-body- (Deng et al 2013), midgut- (Jiang et al 2013), and testis-specific promoters (Xu et al 2015), can also be used to induce FLP gene expression in different tissues of transgenic silkworms and to achieve FLP/FRT system-based genetic mosaics in the soma and germline, as well as chromosome rearrangements, the deletion of large fragments, and other genetic engineering operations in the silkworm genome. In 2003, a binary gene expression system was developed to analyze B. mori gene function using the yeast GAL4 DNA-binding protein and its target cis-element upstream activating sequence (UAS) (Imamura et al 2003).…”