吡哆醛激酶基因在家蚕体内的时空表达特征

Characterization of temporal and spatial expression of pyridoxal kinase gene in Bombyx mori

【目的】了解家蚕Bombyx mori维生素B6关键代谢酶吡哆醛激酶(pyridoxal kinase,PLK)在家蚕不同组织间的表达差异。【方法】原核表达家蚕重组PLK,获得目的蛋白制备多克隆抗体,利用Western blot方法对PLK在家蚕不同发育时期、5龄第3天幼虫不同组织及5龄不同日龄幼虫表皮、头部和马氏管中的表达进行分析;并采用实时荧光定量PCR的方法对PLK基因在家蚕不同组织中的mRNA转录水平进行比较。【结果】PLK在家蚕5龄幼虫的表达水平最高,在5龄第3天幼虫各组织中的表达由高到低依次为:精巢、马氏管、表皮、中肠、头部、卵巢、脂肪体、丝腺;5龄期不同日龄幼虫表皮组织的表达差异最大,头部和马氏管中均为前期表达量稍高于后期。在转录水平方面,精巢的表达量最高,其次是马氏管和头部。【结论】PLK在家蚕不同发育时期及组织中的差异表达进一步证实PLK在家蚕VB6代谢中的生物学功能的重要性。

[Aim] To identify the differential expression of pyridoxal kinase（PLK）,which is a key enzyme related to VB6 metabolism,in different tissues of Bombyx mori.[Methods] The recombinant plasmid was transformed to Escherichia coli Rosetta for induction,expression and purification,and then the polyclonal antibody was prepared.The expression of PLK at different developmental stages,in different tissues of day-3 5th instar larva and in the tissues of cuticle,head and Malpighian tubules on different days of the 5th instar was analyzed by Western blot.The transcription level of PLK gene in different tissues was analyzed by real-time PCR.[Results] The highest expression level of PLK was found in the 5th instar larva.The expression order in various tissues of day-3 5th instar larva from high to low was spermary,Malpighian tubules,cuticle,midgut,head,ovary,fat body and silk glands.The transcriptional analysis of PLK gene on different days of the 5th instar showed that the biggest difference in expression level was in cuticle,and slightly higher on the early days in head and Malpighian tubules.Real-time PCR results showed that PLK gene was highly expressed in spermary,and moderately expressed in Malpighian tubules and head.[Conclusion] The differential expression of PLK gene at different developmental stages and in different tissues of the 5th instar larva further reveals the importance of PLK related to VB6 metabolism in B.mori.