【目的】 昆虫中肠氨肽酶N（Aminopeptidase N，APN）是Bt 杀虫蛋白的重要受体之一，与Bt蛋白的杀虫机制及昆虫对Bt蛋白的抗性密切相关。为阐明稻纵卷叶螟 Cnaphalocrocis medinalis（Guenée）APN基因的功能及明确Bt蛋白对稻纵卷叶螟的毒力机制，本研究系统开展了稻纵卷叶螟中肠APN基因的克隆及时空表达分析。【方法】 通过简并引物 PCR 结合 RACE 技术克隆并获得 4 条稻纵卷叶螟 APN 基因的 cDNA 序列全长，采用实时定量PCR技术研究了APN基因在稻纵卷叶螟不同虫态及幼虫不同组织中的时空表达情况。【结果】 经NCBI同源比对分析，认为这4个基因分属于APN基因家族的不同类别，分别将其命名为CmAPN1（GenBank登录号：HQ853294）、CmAPN2（GenBank登录号：HQ853295）、CmAPN3（GenBank登录号：KJ143755）、CmAPN4（GenBank登录号：HQ853296）。序列分析表明，CmAPN1-4 cDNA序列全长分别为：3 698、3 478、3 150和3 149 bp，开放阅读框分别为：3 045、2 877、3 045和2 862 bp，分别编码 965、958、1 014 和 952 个氨基酸。其推导的氨基酸序列具有鳞翅目昆虫氨肽酶N的典型结构特征，即含有糖基化位点、N-末端信号肽序列、谷氨酸锌化氨肽酶保守结构GAMEN、锌结合位点 HEX2HX18E、C-末端糖基磷脂酰肌醇（GPI）锚信号肽。实时定量研究表明，CmAPNs在幼虫中的表达量显著高于卵、蛹和成虫；在幼虫中，CmAPN1的表达水平明显低于CmAPN2-4，且其在不同龄期中的表达差异显著；CmAPN2-4的表达量随幼虫龄期的增加而增加；CmAPNs 在幼虫肠道组织中的表达量显著高于其它组织器官，且CmAPN1和CmAPN2分别在中肠和后肠中呈现高水平表达，CmAPN3在前、中肠内均高水平表达；但CmAPN4在各个组织器官中的表达均保持较低水平。【结论】 CmAPNs 基因在稻纵卷叶螟的不同虫态和不同组织中呈现了差异显著的时空表达，采用RNA干扰方法进行CmAPNs基因功能研究时，要选择适宜的虫态和虫龄进行干扰。   

    [Objectives] Aminopeptidase N (APN) is an important receptor ofBacillus thuringiensis(Bt) in the midgut of insects that is associated with Bt toxicity and the resistance mechanisms of insect to Bt protein. Cloning and spatio-temporal expression of aminopeptidase N encoding genes inCnaphalocrocis medinalis(Guenée) (Lepidoptera: Pyralidae) were conducted to reveal the function ofCsAPNsand the molecular resistance mechanism ofC. medinalisto Bt protein.[Methods] The full-length cDNA sequence encoding APN was cloned by degenerative primer PCR and RACE techniques. The relative expression levels ofAPNgenes in different developmental stages and various larval tissues ofC. medinaliswere determined by Quantitative Real-time PCR method.[Results] Comparison of sequence identity in NCBI revealed fourAPNgenes belonging to different classes of the APN family were namedCmAPN1(GenBank Accession No. HQ853294),CmAPN2(GenBank Accession No. HQ853295),CmAPN3(GenBank Accession No. KJ143755) andCmAPN4(GenBank Accession No. HQ853296), respectively. The full-length sequences encodingCmAPN1-4were 3 698, 3 478, 3 150 and 3 149 bp, respectively. The open reading frames were 3 045, 2 877, 3 045 and 2 862 bp, which encoded proteins of 965, 958, 1 014 and 952 amino acid residues, respectively. Putative protein sequences included potential glycosylation sites and a zinc metal binding site motif of HEX2HX18E, which is typical of the active sites of the zinc-dependent metalloproteases, and a highly conserved GAMEN motif, which was to form part of the active site. Each sequences had a cleavable N-terminal signal peptide and glycosylphosphatidylinositol (GPI) anchor signal peptide, which is also typical characters of lepidopteran APN proteins. Expression levels ofCmAPNsin larval stages were significantly higher than these in eggs, pupae or adults. During the larval stage, the expression level ofCmAPN1was significantly lower than that ofCmAPN2-4, though it differed significantly among different larval developmental stages. Transcription levels ofCmAPN2-4increased with larval instars.CmAPNspresented significantly different expression levels in different larval tissues, with higher expression in the gut and abdomen and lower expression in the head, thorax, fat body, and entire body minus gut tissues.CmAPN1andCmAPN2showed higher expression in the midgut and hindgut, respectively. Similarly,CmAPN3exhibited significantly high transcription levels in both the foregut and midgut. However, significantly lower expression levels ofCmAPN4were detected among different tissues compared to those ofCmAPN1-3.[Conclusion] CmAPNshave significantly differently spatio-temporal expression patterns in different developmental stages and tissues ofC. medinalis. Therefore, to determine the function ofCmAPNsit is very important to choose the appropriate instar for RNA interference.