《环境昆虫学报》

作者	单位
魏川川，修江帆，胡亚，尚小丽，张迎春，吴建伟	1.贵州医科大学基础医学院，贵阳 550004；2.贵州省疾病预防控制中心，贵阳 550004

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中文摘要:

对家蝇3种丝氨酸蛋白酶抑制剂Serpin基因SP2、SP13和SP16进行克隆、序列分析和时空表达模式检测。运用瑞士生物信息学研究所的蛋白分析专家系统（ExPASy）和美国国家生物技术信息中心（NCBI）等有关的生物信息学分析工具，预测和分析3条Serpin基因编码蛋白质的结构与生物学功能。以RPS18（核糖体S18蛋白）为内参基因，采用实时荧光定量PCR（Real-Time PCR）技术检测3条 Serpin 基因在家蝇不同发育时期，以及家蝇3龄幼虫中脂肪体、唾液腺、中肠、马氏管和体壁的表达特征。SP2、SP13、SP16基因序列的开放阅读框ORF全长分别为1191 bp、1137 bp和1212 bp，分别编码含396、378和403个氨基酸残基的蛋白质，其理论分子量分别为45315.3 Da、43701.5 Da和45011.5 Da，等电点分别为9.01、5.98和6.04，蛋白质的N端都含有一段信号肽，并具有Serpin基因的保守结构域，属于Serpin家族。SP2和SP16在蛋白质的C端含反应中心环（Reactive center loop，RCL)，而SP13 C端不含RCL。时空表达模式分析结果显示，SP2基因在2龄幼虫中的表达量最高，而雄蝇的表达量与卵期相比有所下调；SP13基因在蛹中的表达量最高，1龄幼虫和雌蝇中的表达量与卵期相近；SP16基因在2龄幼虫和3龄幼虫中的表达量最高，雄蝇与雌蝇的表达量与卵期相比有所下调。在家蝇3龄幼虫的各主要组织中，以体壁为参照，SP2基因在唾液腺表达水平最高，其次是脂肪体，中肠和马氏管均低于体壁；SP13基因在马氏管和脂肪体中的表达水平最高，中肠和唾液腺的表达量低于体壁；SP16基因在唾液腺、中肠、脂肪体和马氏管的表达量均低于体壁。推测Serpin基因在家蝇个体发育和免疫调节中起不同作用。

英文摘要:

To probe into the cloning, sequence analysis and temporal expression patterns of three Serpin genes in Musca domestica. SP2, SP13 and SP16 genes were cloned from M.domestica, and their sequences was analyzed. Expert protein analysis system (ExPaSy) of institute of biological information science in Switzerland and national center for biotechnology information (NCBI) from America, and relevant bioinformatics analysis tool were used to predict and analyze the structures and biological functions of threeSerpin genes. The expression of SP2, SP13and SP16gene were detected by Real-time quantitative PCR (Real Time PCR) technique performed in differential developmental stages, and the expression features in fat body, salivary glands, midgut, markov tube and body wall from three instar larvae ofM.domestica, the RPS18 (ribosomal protein S18) as control. The full-length of SP2, SP13 and SP16 genes were 1191 bp, 1137 bp and 1212 bp, and they encoded 396 aa、378 aa and 403 aa respectively. The predicted molecular weight of SP2, SP13 and SP16were 45315.3 Da， 43701.5 Da and 45011.5 Da, and isoelectric point were 9.01, 5.98 and 6.04,respectively. All of them contained a signal peptide, and the C terminal of SP2 and SP16contained a reactive center loop, while SP13 had not. The spatial and temporal expression patterns showed that the expression of SP2 gene was highest in the second instar larvae, but that was reduced compared with the egg stage, while the expression ofSP13 was highest in the pupa, and the expression of first instar larvae and female similar with the eggs. Besides, the expression of SP16 gene was highest in the second instar larvae and third instar larvae, but the expression of male and female were reduced compared to the egg stage. The expression of these genes in the major organizations of third instar larvae in M.domestica showed that the expression ofSP2 was highest in the salivary glands, then in fat body, midgut and Markov tube were lower than that of body wall, the expression in body wall as control; while the expression of SP13 gene was highest in the Markov tube and fat body, then was body wall, midgut and salivary glands. At last, the expression of SP16 gene was highest in the body wall, then was fat body, salivary glands, midgut and Markov tube in that order. The results indicated that these genes play different roles in the individual development and immune regulation of M.domestica.

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