家蝇Ⅲ龄幼虫消化系统内源性β-葡萄糖苷酶的组织定位和表达差异研究  

Tissue Localization and Expression Difference of Endogenousβ-glucosidase in Digestive System of Musca domestica Third Instar Larvae

作　　者：胡蓉[1] 张姝[1] 吴建伟[1] 国果[1] 付萍[1] 

机构地区：[1]贵阳医学院基础医学院,贵阳550004

出　　处：《中国寄生虫学与寄生虫病杂志》2013年第4期256-261,共6页Chinese Journal of Parasitology and Parasitic Diseases

基　　金：国家科技支撑计划项目(No.2011BAC06B12);国家自然科学基金(No.30960343);国家教育部博士点专项基金(No.2008-220);教育部高等学校博士学科点专项科研基金新教师类课题(No.20105215120001);贵州省教育厅培育项目(No.2009-0137)~~

摘　　要：目的以家蝇Ⅲ龄幼虫为研究对象,探讨家蝇(Musca domestica)消化系统内源性β-葡萄糖苷酶(β-glucosidase,BG)的组织分布、表达差异和器官功能定位。方法用原位杂交鉴定家蝇β-葡萄糖苷酶mRNA的组织定位。免疫组织化学法鉴定纤维素酶的组织定位。分离家蝇Ⅲ龄幼虫消化系统组织,用3,5-二硝基水杨酸(DNS)法测定β-葡萄糖苷酶的活性,实时荧光定量PCR法检测家蝇β-葡萄糖苷酶mRNA在消化系统各器官中的表达差异。结果原位杂交显示,中肠、前肠和唾液腺上皮细胞是家蝇β-葡萄糖苷酶mRNA表达的部位。免疫组织化学染色显示,唾液腺、前肠和中肠上皮细胞是家蝇幼虫产生和分泌纤维素酶的主要部位。DNS法测定结果显示,家蝇幼虫唾液腺、前肠、中肠和后肠的β-葡萄糖苷酶活性分别为(0.80±0.06)、(0.38±0.02)、(1.20±0.05)和(0.26±0.02)IU/mg,各部位间酶活性差异有统计意义(P<0.05);中肠的酶活性较高,占总酶活性的(45.45±1.27)%,后肠的酶活性最低,为(9.85±0.88)%。实时荧光定量PCR显示,家蝇β-葡萄糖苷酶mRNA只在前肠、中肠和唾液腺中表达,中肠的表达量约为前肠的5倍,唾液腺的表达量约为前肠的3倍,3者表达量差异有统计学意义(P<0.05),而后肠未见该基因的表达。结论家蝇Ⅲ龄幼虫前肠、中肠和唾液腺均能分泌内源性β-葡萄糖苷酶,唾液腺和中肠为主要分泌器官。Objective To study the tissue localization and expression difference of endogenous β-glucosidase in digestive system of Musca domestica third instar larvae. Methods The digestive system of the 3rd instar larvae of Musca domestic was taken for the below tests. Tissue localization of endogenous β-glucosidase mRNA was identified by in situ hybridization. Cellulase was localized by immunohistochemistry. The enzymatic activity of β-glucosidase was measured by 3, 5-dinitrosalicylic acid（DNS） assay. The relative mRNA expression levels of M. domestica β-glucosidase gene in these organs were determined by RT-PCR. Results β-glucosidase mRNA, with in situ hybridization, was shown in the epithelial cells of midgut, salivary glands and foregut of the larvae. The immunohistochemical analysis on larvae tissues revealed that cellulase was produced and secreted by the epithelial cells of the midgut, salivary glands and foregut. β-glucosidase activity in salivary glands, foregut, midgut, and hindgut was（0.80＋0.06）, （0.38±0.02）, （1.20±0.05） and（0.26±0.02） IU/mg, respectively. There was significant difference in β-glucosidase activity among these digestive organs （P〈 0.05）. The activity level of β-glucosidase was highest in midgut [（45.45±1.27）%], and lowest in hindgut[（9.85±0.88）%]. However, β-glucosidase gene were only expressed in the salivary gland, foregut and midgut, Significant differences in gene expression level ofβ-glucosidase was found among these organs（P〈0.05）. The relative expression quantity of β-glucosidase gene in midgut and salivary glands were 5 and 3 times higher than that in foregut. Conclusion The endogenous β-glucosidase gene is expressed in the foregut, midgut and salivary glands. The midgut and salivary glands of Musca domestiea 3rd instar larvae are the primary organs of this enzyme secretion.

关 键 词：Β-葡萄糖苷酶 消化系统 家蝇 

分 类 号：R384.21[医药卫生—医学寄生虫学]