Phylogenetics of hexokinase gene and expression characteristics of its multiple transcripts in the early embryonic stage of Bombyx mori

【Objective】To clarify the expression characteristics of the different transcripts of the hexokinase gene(BmHK) in the early embryonic development stage of bivoltine silkworm(Bombyx mori), and in order to provide a theoretical basis for further exploring the biological functions of each transcript of the BmHK gene and revealing the mechanism of action in the early embryonic development of silkworm.【Method】The HK amino acid sequences of 12 species of insects including B. mori were obtained and downloaded from NCBI, and a phylogenetic tree based on the similarity of HK amino acid sequences was constructed using the neighbor-joining method(NJ) of MEGA 7.0. The non-diapause-destined eggs(ND), diapause-destined eggs(DD) and immediately acid-treated eggs(IA) were prepared using Qiufeng activated hibernating eggs(C-2 embryos) of the silkworm bivoltine strain. Real-time fluorescence quantitative PCR was used to detect the expression characteristics of each transcript during the early embryonic development of silkworm.【Result】The BmHK gene belonged to the HSP70-actin family, and there were 4 different transcripts with lengths of 4610, 4608, 2818, and1520 bp, respectively named BmHK-a, BmHK-b, BmHK-c and BmHK-d. BmHK-a showed an overall trend of increasing first and then decreased during the early embryonic development of the three kinds of silkworm eggs. At each time point, it was expressed as IA silkworm eggs>ND silkworm eggs>DD silkworm eggs, in IA silkworm eggs the relative expression of BmHK-a reached a peak at 2 h of development, and reached a peak at 48 h of development in ND silkworm eggs and DD silkworm eggs. After that, its expression was rapidly down-regulated and remained at a low level until 96 h of development;expression level of BmHK-b in silkworm eggs in three species was as follows:IA silkworm eggs>ND silkworm eggs>DD silkworm eggs. The relative expression levels of BmHK-b in the three silkworm eggs had basically the same trend, and they all showed a gradual upward trend with the development time. The expression of Bm HK-c in the three silkworm eggs was greatly different. The expression level in the first-laying silkworm eggs was already high, and it increased rapidly after fertilization. The relative expression level of BmHK-c in the three silkworm eggs at 72 h after development increased again;the relative expression of BmHK-d in the three silkworm eggs showed an overall trend of first increasing and then decreasing, and at each time point it was ND silkworm eggs>IA silkworm eggs>DD silkworm eggs, but the peak of BmHK-d expression in IA silkworm eggs appeared earlier than DD silkworm eggs and ND silkworm eggs.【Conclusion】BmHK-a and BmHK-d mainly play an important role in the early embryonic development of the silkworm(3 d after laying eggs or soaking in acid);although BmHK-b is also involved in the early embryonic development of the silkworm, it is not the main sugar metabolism related enzymes;BmHK-c is closely related to the early embryonic development of the silkworm, and provides energy for embryonic development through glycolysis.