家蚕酪氨酸羟化酶基因BmTh的表达及功能

Expression profile and function of tyrosine hydroxylase gene BmTh in the silkworm,Bombyx mori

酪氨酸羟化酶作为儿茶酚胺合成的限速酶,广泛存在于昆虫、哺乳动物和人类中,是其新陈代谢不可缺少的酶类。在其他昆虫中,酪氨酸羟化酶参与了黑色素的合成,并在昆虫外骨骼的硬化过程中发挥关键作用。为了研究家蚕Bombyxmori酪氨酸羟化酶基因的生理生化功能,本文对其基因结构、表达特征及功能进行了研究。基于家蚕基因组和基因芯片数据的生物信息学分析表明,BmTh位于家蚕1号染色体上,含有8个外显子,编码561个氨基酸。基因芯片数据显示在家蚕5龄第3天的头部和体壁组织中的表达量较高,RT-PCR验证结果与此一致。利用石蜡组织切片材料和RNA探针对BmTh进行表达定位,原位杂交结果显示在家蚕头部边缘和体壁上有明显的杂交信号。在幼虫发育至熟蚕时注射酪氨酸羟化酶抑制剂3-indole-L-tyrosine(3-IT),20mmol/L的浓度对幼虫几乎没有影响,50mmol/L的浓度导致幼虫变态不完全和化蛹困难,100mmol/L的浓度使幼虫致死且体色变黑。结果提示,BmTh对家蚕变态发育起重要作用,是家蚕正常发育不可缺少的关键基因。

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.