家蚕谷胱甘肽-S-转移酶E4基因的组织表达和序列特征及重组蛋白的酶活性分析

Tissue-specific Expression,Sequence Characteristics and Enzyme Activity Assay of Recombinant Protein of Bombyx mori Glutathione S-transferase E4 Gene

昆虫体内的谷胱甘肽-S-转移酶(GSTs)主要行使对异生或内源有毒物质进行代谢解毒的功能。选择家蚕谷胱甘肽-S-转移酶E4的编码基因Bmgste4为靶标,研究其组织表达谱、序列结构特征及原核表达重组蛋白的酶活性。RT-PCR检测不同组织表达特征的结果显示,Bmgste4基因在家蚕5龄第3天幼虫的头部、触角、下颚须、表皮、脂肪体、丝腺以及雄性个体的马氏管和雌性个体的卵巢中均有表达,而在血细胞、中肠以及雄性个体的精巢和雌性个体的马氏管中不表达。多序列比对结果显示BmGSTE4具有完整和保守的GST二级结构特征,N端氨基酸残基保守,特别是谷胱甘肽结合位点。构建插入Bmgste4基因开放阅读框片段的重组表达质粒p28-Bmgste4,并转化E.coli Rosetta(DE3)感受态细胞进行原核表达后,利用分光光度法测定重组蛋白对通用底物1-氯-2,4-二硝基苯的酶活参数:Km=0.58 mmol/L,Vmax=24.55μmol/(mg.min)。这些结果为进一步研究家蚕体内解毒和抗药性机制以及开发鳞翅目害虫新型生物防治药剂提供了基础信息。

Insect glutathione Stransferases （GSTs） are mainly involved in detoxification of xenobiotics and/or endobiot ics toxic substances. We selected Bmgste4 gene which encodes silkworm （ Bombyx mori） GST E4 as the target to study its tissue expression profile, sequence characteristics and enzyme activity of the recombinant protein. RTPCR analysis on expression profile in various tissues of day 3 silkworm larvae of the 5th instar indicated that Brngste4 was expressed in head, antenna, maxillary palpus, epidermis, fat body, silk gland, Malpighian tubule of male larvae and ovary of female larvae but not expressed in haemocyte, midgut, testis of male larvae and Malpighian tubule of female larvae. Multiple sequence alignment showed that the GST secondary structural features of BmGSTD4 are complete and conservative. Its Nterminal amino acid residues are highly conservative, especially at the glutathione binding site. Recombinant protein expression plasmid p28Bmgste4 containing open reading fragment of Bmgste4 gene was constructed and transformed into competent E. coil Rosetta （DE3） for pro karyotic expression. Enzyme activity of the recombinant protein towards classic substrate 1chloro2,4dinitrobenzene was assayed by spectrophotography. The obtained enzymatic parameters were Km =0.58 mmoVL and Vmax = 24.55 μmol/（mg.min）. These results provide fundamental information for further study on detoxification and insecticide resistance of Bombyx mori and on development of novel agents for controlling lepidopteran pests.