Abstract: 【Aim】 The main aim of this research was to clone and align the full-length cDNA and promoter sequences of desat4 gene from silkworms, Bombyx mori and B. mandarina, and construct prokaryotic expression vector to obtain a membrane protein Desat4 for further functional study. 【Methods】 Full-length cDNA of the desat4 gene from silkworm was obtained through RACE technique, the encoded protein was expressed in Escherichia coli expression system, and its promoter sequences were cloned based on genome sequences of silkworm. 【Results】 The full-length cDNA of the desat4 gene from B. mori and B. mandarina was 1 717 bp and 1 718 bp, respectively. Their open reading frame (ORF) is 1 059 bp in length and encodes 352 amino acid residues with four transmembrane helixes and three conserved histidine clusters, which are essential for desaturase catalytic activity. The deduced amino acid sequence shares 88.9% similarity to that of the fatty acid desaturase MsexKPSE (GenBank no. CAJ27975) of Manduca sexta. There is no typical TATA-box in promoter sequences, but there is a transcriptional initiator as well as other transcription factor binding sites, including HSF, NIT2, CdxA and so on. The membrane protein Desat4 was expressed in E. coli expression system and solubilized with mild detergent DDM. 【Conclusion】 The full-length cDNA and promoter sequences of desat4 gene from B. mori and B. mandarina were successfully cloned and comparatively analyzed. The membrane protein was expressed in vitro by pET system, thus providing a foundation for further functional study.

Key words: Bombyx mori, Bombyx mandarina, fatty acid desaturase, fulllength cDNA, promoter, prokaryotic expression