【摘要】 在昆虫的免疫防御系统中,模式识别受体介导的免疫反应起着主导性作用。采用RACE方法克隆了柞蚕的模式识别受体——β-1,3-葡聚糖识别蛋白的基因ApβGRP（GenBank登录号:JN880424）。序列分析表明:ApβGRP含有1 470 bp的开放阅读框,编码490个氨基酸;ApβGRP属于分泌型蛋白质,N端有17个氨基酸组成的信号肽,具有βGRP保守性的糖苷水解酶活性结构域（Glyco_hydro₁6）。系统进化分析显示ApβGRP与家蚕的βGRP-3属于同一个分支,序列相似度为71%。利用实时定量PCR方法检测柞蚕蛹经4种微生物（革兰阳性菌Bacillus subtilis、革兰阴性菌Escherichia coli Top10、病毒Antheraea pernyi nucleopolyhedrovirus、真菌Saccharomyces cerevisiae W303-1A）诱导处理后,ApβGRP在蛹体表皮、脂肪体和血淋巴中均明显上调表达,但在生殖腺内的表达则无明显变化,该基因在中肠内仅被革兰阳性菌及真菌诱导上调表达,并且该基因对真菌的应答表达最显著,显示ApβGRP经不同微生物诱导后的表达水平以及在蛹体不同组织中的表达水平有明显差异。推测ApβGRP因具有糖苷水解酶活性结构域能水解病原体的多糖,所以可以通过与微生物表面相关分子模式的结合而识别多种微生物,并调控相关的免疫通路,在柞蚕免疫系统中发挥作用。更多还原

【Abstract】 Immunoreaction mediated by pattern recognition receptors plays a predominant role in insect immune defense system.In this paper,we cloned the Antheraea pernyi pattern recognition receptor protein（β-1,3-glucan recognition protein） gene Ap β GRP（GenBank accession No.JN880424） by RACE technology.Sequence analysis indicated that Ap β GRP has an open reading frame of 1 470 bp and codes for 490 amino acids.ApβGRP is a secreted protein with a signal peptide consisting of 17 amino acids at the N-terminus and contains a conservative βGRP domain with glycosidic hydrolase activity（Glyco_hydro₁6）.Phylogenetic analysis showed that ApβGBP and Bombyx mori βGBP-3 belonged to the same clade,and had a sequence identity of 71%.Real-time quantitative PCR was conducted to detect the expression pattern of Ap β GRP in different tissues of silkworm after infection by 4 different microorganisms,namely Gram-positive bacteria Bacillus subtilis,Gram-negative bacteria Escherichia coli Top10,Antheraea pernyi nucleopolyhedrovirus,and Saccharomyces cerevisiae W303-1A.It was found that the expression level of Ap β GRP was obviously up-regulated in epidermis,fat body,hemocyte,while no obvious change was observed in gonad.Meanwhile,Ap β GRP could be up-regulated in midgut under infection of Gram-positive bacteria and fungus,and the response of Ap β GRP to fungus was the most significant,all of which showed that the expression level of Ap β GRP had remarkable difference in various tissues of silkworm after infection by different microorganisms.It is considered that,because ApβGRP possesses the domain with glycosidic hydrolase activity which enables it to hydrolyze pathogenic polysaccharides,ApβGRP recognizes different microorganisms through binding with relevant molecular patterns in the surfaces of the microorganisms,and then regulates the related immune pathways to perform its function in the immune system of Antheraea pernyi.更多还原