家蚕前部丝腺特异表皮蛋白Bm11721的鉴定及表达

Identification and expression patterns of anterior silk gland specific cuticle protein Bm11721 in the silkworm (Bombyx mori)

摘要:

家蚕的丝腺是其丝蛋白合成和分泌的器官，根据其形态和功能的不同分为前部、中部和后部丝腺，前部丝腺不具有合成丝蛋白的能力，是丝蛋白构象发生转变的场所。剪切力在丝蛋白构象转变中起到重要的作用，其在家蚕前部丝腺主要由前部丝腺逐渐变细的管腔结构和富含几丁质及表皮蛋白的坚硬的内壁提供。鉴定家蚕前部丝腺新的几丁质结合蛋白，并调查其在家蚕幼虫不同组织的表达特征。通过几丁质亲和层析的方法在前部丝腺筛选并鉴定到一个新的具有几丁质结合功能的表皮蛋白Bm11721，其编码基因编号为BGIBMGA011721 (GenBank Accession No. NM-001173285.1)。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Bm11721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Bm11721均只在前部丝腺特异表达，且Bm11721蛋白在5龄期的前部丝腺中恒定表达。免疫荧光定位结果显示Bm11721蛋白定位在前部丝腺的内膜中，推测其可能与前部丝腺的机械硬度有关，为丝蛋白的构象转变提供剪切力。

Abstract:

The silk gland of silkworm is the organ of silk protein synthesis and secretion. According to the morphological and functional differences, silk gland can be divided into anterior silk gland (ASG), middle silk gland (MSG) and posterior silk gland (PSG). ASG is the place for silk proteins conformation changes although it cannot synthetize silk proteins. ASG has narrow luminal structures and rigid wall which consists of chitin and cuticle proteins so that it can provide the shearing force which plays an important role in the silk protein conformation changes. The objective of this study is to identify the new chitin binding proteins in ASG of silkworm (Bombyx mori), and to analyze their expression patterns in different tissues. We identified a cuticle protein with chitin binding domain Bm11721 (GenBank Accession No. NM-001173285.1) by chitin affinity chromatography column. We also expressed the recombinant protein as inclusion body using the prokaryotic expression system, and then successfully purified the recombinant protein by nickel affinity chromatography column to generate the polyclonal antibodies. The expression patterns analysis in various tissues showed that both in transcriptional and protein levels Bm11721 was specifically expressed in ASG. Furthermore, the expression level of Bm11721 protein was unchanged during the 5th instar. Immunofluorescence analysis revealed that Bm11721 was located in the ASG inner membrane. It is proposed that Bm11721 is a component of inner membrane and probably provides the shearing force for conformational changes.