家蚕保幼激素结合蛋白Bmtol基因的克隆及功能分析

Expression and characterization of juvenile hormone binding protein Bmtol gene in silkworm, Bombyx mori

摘要:

家蚕头部是一个神经中枢和感受的器官，其头部含有触角和感觉毛，感受外界的信号，并将外界信号传送到大脑进行反应。保幼激素主要是由咽侧体合成和分泌的，而保幼激素结合蛋白是保幼激素转运和发挥功能的载体，在昆虫体内具有极其重要的功能。文中通过SilkDB和NCBI数据库筛选并鉴定到一个新的具有保幼激素结合蛋白家族保守结构的蛋白BmTOL，其编码基因编号为BGIBMGA003404 (GenBank登录号：KY681053)。利用原核表达系统成功表达了该蛋白，通过Ni-NTA亲和层析的方法获得了BmTOL的重组蛋白并制备了多克隆抗体。组织表达分析发现无论是转录水平还是蛋白水平BmTOL在头部都是高量表达，且Bmtol基因在起蚕时表达量较高，在5龄和蛹期表达量较低，而在化蛾后表达量又开始上调。免疫组化结果显示BmTOL蛋白定位在头部的皮层、触角和脑中，推测其可能与头部信息传递有关，为家蚕的生长发育和行为调控提供重要的信息来源。

Abstract:

The head of the silkworm is a nerve center and a sense organ, contains antennaes and sensory hair, feels the outside signal, and responds to the external signal delivered to the brain. Juvenile hormone is mainly synthesized and secreted by corpora allata, and it needs to be played with the aid of the hormone binding protein, because the juvenile hormone binding protein is the carrier of juvenile hormone transport and plays a functional in vivo, they have an extremely important function in insects. The objective of this study is to screened and identify a novel BmTOL proteins that it has a conserved structure of the juvenile hormone binding protein family by SilkDB and NCBI database. Its coding gene number is BGIBMGA003404 (GenBank Accession No. KY681053). We also expressed the recombinant protein using the prokaryotic expression system, and then successfully purified the recombinant protein by Ni-NTA chromatography column to generate the polyclonal antibodies. The expression patterns analysis in various tissues showed that both in transcriptional and protein levels Bmtol was higher expressed in head. Furthermore, the expression level of Bmtol gene was higher in newly exuviated silkworm, and expression level of Bmtol gene was lower from at 3 days 5th instar to 7 days pupa, began to increase after the moth. Immunohistochemistry showed that BmTOL protein was localized in the cortex, antennaes and brain of the head, It may be related to the information transmission of the head, and provides an important source of information for the growth and development of silkworm.