Molecular cloning and sequencing of an insect intestinal mucin cDNA from spodoptera exigua

Chinese. 【目的】围食膜（peritrophic membrane,PM）是衬托于昆虫中肠内的一种无脊椎动物特有的几丁质―蛋白复合结构,它能够促进食物消化,保护消化道免受微生物入侵。昆虫肠粘蛋白（ⅡM）是其重要组分之一。因此,作为中肠防御系统的PM已成为害虫生物防治的新靶标。分离和鉴定甜菜夜蛾肠粘蛋白基因cDNA。【方法】以甜菜夜蛾（Spodoptera exigua）中肠为材料,依据stratagene公司试剂盒方法,构建甜菜夜蛾中肠cDNA表达文库。依据现代免疫学原理,利用粉纹夜蛾肠粘蛋白特异性多克隆抗体（anti-ⅡMantiserum）筛选该文库。【结果】cDNA表达文库滴度为8.51×106pfu/ml,重组率为99.97%。筛选文库得到一个编码甜菜夜蛾肠粘蛋白的cDNA克隆SeⅡM-8。核酸序列分析显示,该cDNA长为2234bp,在polyA末端上游24bp处有一个多聚腺苷酸信号序列AAATTA。最长读码框（ORF）编码714个氨基酸,与粘虫肠粘蛋白（Mamestra configurata intestinal mucin）序列相似性为42%。结构域分析表明,它具有5个几丁质结合功能域;一个富含苏氨酸类粘蛋白结构域,重复序列为TTTQAPTTT,高度O-联糖基化;一个天冬氨酸富集区,序列DDDKPGCNGNCPE重复达12次,该区域在已知的昆虫肠粘蛋白中属首次发现。与已知肠粘蛋白比较,推测其5′端还应包括信号肽序列和几丁质结合功能域的未知序列。SeⅡM-8基因在GenBank登录号为EU047712。【结论】从本研究构建的高质量甜菜夜蛾中肠cDNA表达文库中筛选得到一个新的围食膜蛋白基因SeⅡM-8。

English. [Objective] The insect midgut is generally lined with a unique chitin/protein structure, the peritrophic membrane （PM）. PM facilitates the digestive process, and protects invertebrate digestive tracts from microbial infections. The insect intestinal mucin （IIM） is a major protein constitute of the PM. Therefore, PM could be potential target for developing pest control technique and the isolation and identification of IIM gene is necessary. [ Method ] To clone and sequence the cDNA for SeIIM, a Spodoptera exigua larval midgut cDNA expression library was constructed and screened with an anti-T.ni IIM antiserum. [ Result ] The original cDNA expression library contained 8.51 × 10. recombinants and the recombination rate was 99.97%. The titer of amplified library is 2.41 - 109 pfu/ml. The average length of the inserted fragments was 1.9 kb by enzyme digestion. A positive cDNA clone for IIM, named as SeIIM-8, was identified and sequenced. The cDNA is 2 234 bp, and the longest open reading frame codes for 714 amino acids. The similarity to Mamestra configurata intestinal mucin is 42% in amino acids sequence, It contains five chitin binding domains and one threonine-rich mucin domain. One tandem repeating unit TTTQAPTTT repeats 6 times among the mucin domain which is potential O-glycosylation sites. One aspartic acid-rich region contains one repeating unit, DDDKPGCNGNCPE repeats 12 times among the aspartic acid-rich region that distinguished llMs from other insects. It is referred that there are one signal peptide and unknown CBDs in the 5＇ end of SeIIM-8. The accession number in GenBank for SeIIM-8 is EU047712. [ Conclusion ] A cDNA expression library with high quality from S. exigua midgut is constructed and a novel protein which named as SeIIM-8 coding for IIM is identified in this study.