关键词: 琥珀蚕, 孵化酶, RACE, 表达谱, 启动子活性, BmN细胞

Abstract: 【Aim】 Antheraea assama has the typical characteristics of wild silkworms, with its eggs not uniformly hatched, which seriously affects its largescale indoor rearing. This study aims to explore the characteristics of the hatching enzyme gene playing a key role in the egg hatching of A. assamensis and its promoter sequence, and to lay the foundation for further selection of appropriate inhibitors or accelerators to regulate the egg hatching of A. assamensis. 【Methods】 The full-length cDNA sequence of the hatching enzyme gene was cloned from A. assamensis by RACE, and the sequence was analyzed by bioinformatics method. qRT-PCR was used to detect the expression profiles of the hatching enzyme gene in eggs of different days after laying, and in different tissues (silk gland, Malpighian tubules, head, midgut, fat body, epidermis, blood, testis and ovary) of the day-3 and day-4 5th instar larvae of A. assamensis. The promoter sequence of the hatching enzyme gene was cloned from A. assamensis by chromosome walking, and the promoter activity was detected by constructing the insect cell recombinant expression vector and transfecting the BmN cells of Bombyx mori. 【Results】 The full-length cDNA sequence of the hatching enzyme gene AaHE (GenBank accession no.: KT336227.1) was obtained from A. assamensis. It is 993 bp in length, encoding 294 amino acids with the predicted molecular weight of 33.7 kD and the theoretical isoelectric point of 5.17. The amino acid sequence of AaHE contains a signal peptide and a ZnMc domain, and AaHE belongs to a group of zinc-dependent proteolytic enzymes with a HExxH zinc-binding site. The members of this enzyme group are both peptidase and digestive enzyme. AaHE was specifically and highly expressed in the eggs before hatching and the midgut of the 5th instar larva of A. assamensis, being consistent with the properties of peptidase and digestive enzyme. There are multiple transcription factor binding sites in the core region of AaHE promoter, which may be related to the transcription factor’s involvement in regulating the expression of AaHE. Promoter activity analysis showed that the AaHE promoter could initiate the expression of EGFP in the BmN cells of B. mori and had obvious promoter activity. 【Conclusion】 AaHE is a zinc-dependent proteolytic enzyme with multiple transcription factor binding sites in the core region of its promoter. This study provides references for choosing suitable inhibitors or accelerators to regulate the egg hatching of A. assama.

Key words: Antheraea assama, hatching enzyme, RACE, expression profile, promoter activity, BmN cell