关键词: 家蚕, 酪氨酸羟化酶基因, 生物信息学分析, 组织定位, 基因芯片, 原位杂交

Abstract:

As a key enzyme of catecholamine synthesis, tyrosine hydroxylase is an indispensable enzyme of metabolism in insects, mammals and human. Tyrosine hydroxylase involves in the synthesis of melanin, affects the body color and insect exoskeleton hardening. In order to explore the physiological and biochemical function of tyrosine hydroxylase, in this study, the structure, expression profile and function of tyrosine hydroxylase gene were studied in the silkworm, Bombyx mori, using the combined methods of bioinformatic analysis and experiments. The bioinformatic analysis based on the silkworm genome database and microarray database indicated that the silkworm tyrosine hydroxylase gene BmTh is located on chromosome 1. BmTh includes 8 exons and encodes a protein with 561 amino acid residues. Microarray data in different tissues showed that it has significant expression signals in head and epidermis of day-3 5th instar larvae, which was verified by RT-PCR. In situ hybridization analysis showed that there was remarkable signal on the rim of head and epidermis with paraffin tissue section and RNA probe. Through injecting an inhibitor of tyrosine hydroxylase (3-indole-L-tyrosine, 3-IT) at different concentrations into silkworm larva at the mounting stage, we found that 20 mmol/L 3-IT had no influence on the larval development, 50 mmol/L 3-IT could handicap the larval metamorphosis and pupation, and 100 mmol/L 3-IT could cause the larval death and turn the larval body color into black. These results suggest that tyrosine hydroxylase plays an important role in the processes of metamorphosis and it is an indispensable key enzyme for normal development in the silkworm.

Key words: Bombyx mori, tyrosine hydroxylase gene, bioinformatic analysis, tissue localization, microarray, in situ hybridization