柞蚕滞育关联蛋白基因dap2的表达特征及分子特性

Expression Features and Molecular Properties of Diapause-associated Protein Gene dap2 of Antheraea Pernyi

滞育关联蛋白(DAP)在昆虫滞育进程中可能发挥重要作用。从NCBI获取柞蚕滞育关联蛋白基因2(Antheraea pernyi diapause-associated protein 2,dap2)的序列,序列分析表明该基因的cDNA全长1 187bp,ORF为874bp,编码278个氨基酸。NCBI数据库氨基酸序列比对,DAP2与烟草天蛾和家蚕的眼色素结合蛋白(omminbinding protein,OBP)同源性分别达到57%和58%。通过荧光定量PCR和Western blotting方法,分别检测柞蚕不同发育时期和五龄幼虫不同组织中dap2的转录表达水平,结果表明dap2在滞育蛹中转录和表达水平最高;在5龄幼虫不同组织中的分析表明其转录水平在脂肪体和血淋巴中最高,在翻译水平上血淋巴中表达最高。分离纯化后,对天然DAP2蛋白分子特性进行初步研究,吸收光谱分析不能表明其携带色素,去糖基化修饰分析表明其为糖蛋白,双向电泳表明其可能存在三种不同程度的翻译后修饰,推测其可能是柞蚕的一种眼色素结合蛋白(ommin-binding protein,OBP)。

Diapause-associated protein（DAP）might play an important role in the diapause process of insects.The sequence of Antheraea pernyi diapause-associated protein 2,dap2 is obtained from NCBI.Sequence analysis shows that the overall length of cDNA of this gene is 1187 bp and that of ORF is 874 bp and 278 amino acids are encoded.According to NCBI database amino acid sequence alignment,the homology of DAP2 with ommin-binding protein（OBP）of tobacco hornworm and silkworm respectively reaches57%and 58%.The transcriptional expression level of dap2 mRNA in different development stages of antheraea pernyi and different tissues of five-instar larvae is respectively tested with fluorescent quantitation PCR and western blotting method.The result shows that dap2 has the highest transcriptional and expression level in diapause pupae.The analysis in different tissues of five-instar larvae shows that its transcriptional level is highest in fat body and hemolymph and its translational level is highest in hemolymph.Preliminary study is conducted on molecular characteristics of natural ApDAP2 protein after separation and purification.Absorption spectrum analysis cannot show that it carries pigment.Deglycosylation modification analysis shows that it is glycoprotein and dimensional electrophoresis shows that there might be posttranslational modification at three different degrees.It might be an ommin-binding protein（OBP）of antheraea pernyi.