关键词: 家蚕, 精巢发育, 蜕皮激素, FoxL2-2, 转录调控

Abstract: 【Aim】 To clarify the molecular mechanism of 20-hydroxyecdysone (20E) in regulating the expression of the transcription factor gene BmFoxL2-2 of Bombyx mori, and analyze the expression pattern of SlFoxL2-2 in the testis of Spodoptera litura. 【Methods】 The 2 500 bp promoter sequence upstream of the initiation codon of BmFoxL2-2 was cloned from the testis genomic DNA of the 5th instar larva of B. mori by PCR and the potential cis-regulatory elements (CREs) were predicted by bioinformatics tools. pEGFP-BmBRC-Z1, pEGFP-BmBRC-Z2 and pEGFP-BmBRC-Z4 transcription factor expression plasmids and pGL3-BmFoxL2-2 plasmid were co-transfected into the BmN cells and the dual luciferase activity assay was performed to analyze the effects of BmBRCs on the promoter activity of BmFoxL2-2. The expression level of BmFoxL2-2 in the BmN cells at 4 h after treatment with 1 μmol/L 20E was determined by qRT-PCR, and the expression levels of BmBrc-z1 and BmBrc-z4 were detected as the control. The expression levels of BmBrc-z1 and BmBrc-z4 in the testis at different developmental stages (larva, prepupa, pupa and adult), and in the testis and ovary of the 5th instar larva and 3-day-old pupa of B. mori were determined by qRT-PCR. SlFoxL2-2 in the genome database of S. litura was identified by bioinformatics and the expression levels of SlFoxL2-2 in the testis and ovary of the 6th instar larva and adult, different tissues (fat body, midgut, haemolymph, head, cuticle and testis) of the 6th instar larva, the testis at different developmental stages, and in the testis, sperm and testis membrane of the 6th instar larva were determined by qRT-PCR. 【Results】 The about 2 500 bp promoter sequence upstream of the initiation codon of BmFoxL2-2 (Gene ID: 101735653) of B. mori was obtained, and 13 potential CREs of BRC were predicted. BmBRC-Z1 and BmBRC-Z4 significantly up-regulated the promoter activity of BmFoxL2-2, and the expression levels of BmFoxL2-2, BmBrc-z1 and BmBrc-z4 in the BmN cells at 4 h after treatment with 20E were also significantly up-regulated as compared with those of the control group. BmBrc-z1 and BmBrc-z4 were expressed in the testis of larva, prepupa, pupa and adult of B. mori. The expression levels of BmBrc-z1 and BmBrc-z4 in the testis of the 5th instar larva and 3-day-old pupa were significantly higher than those in the ovary. The expression level of SlFoxL2-2 of S. litura was high in the testis among different tissues, and was significantly higher in the testis and sperm than in the testis membrane of S. litura larvae, suggesting that SlFoxL2-2 also functions in the development of testis, especially in the spermatogenesis. 【Conclusion】 The expression of BmFoxL2-2 is regulated by BmBRC-Z1 and BmBRC-Z4 through 20E in B. mori, and the function of SlFoxL2-2 in the development of testis may be conserved in lepidopteran insects.

Key words: Bombyx mori, testis development, ecdysteroid, FoxL2-2, transcriptional regulation