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昆虫中肠氨肽酶N是Bt毒素的重要受体之一,与Bt毒素的杀虫机制及昆虫Bt抗性的产生密切相关。本研究通过简并引物PCR结合RACE技术克隆并获得大螟Sesamia inferensBt受体蛋白——氨肽酶N(aminopeptidase N,APN)基因的cDNA序列全长,经NCBI同源比对分析,认为该基因为APN3基因,并将其命名为SiAPN3(GenBank登录号为HQ636624)。序列分析表明,该基因的cDNA序列全长为3411bp,开放阅读框为3018bp,编码1006个氨基酸;预测蛋白质分子量为114kD,等电点为4.95;其推导的氨基酸序列中具有鳞翅目昆虫氨肽酶N典型的结构特征,即含有1个N-和12个O-连接的糖基化位点,N-末端具有18个氨基酸的剪切信号肽,谷氨酸锌化氨肽酶保守结构GAMEN,锌结合位点HEXXHX18E,C-末端具有22个氨基酸的糖基磷脂酰肌醇(GPI)锚信号肽。采用实时定量PCR研究了SiAPN3在大螟4龄幼虫肠道不同部位和幼虫不同龄期的转录表达谱。结果表明,该基因在幼虫中肠中的表达量最高,其次为后肠,前肠中表达量最低,且中肠和后肠中的表达量显著高于前肠(P<0.05);SiAPN3在3龄幼虫中的表达量最高,1龄幼虫中最低,虽然3、4日龄之间的表达量没有显著差异,但二者均显著高于其他日龄,1,2和5日龄之间不存在显著差异(P>0.05)。该研究为阐明APN基因的功能及大螟对Bt抗性产生的分子机制奠定了基础。
Insect midgut aminopeptidase N is one of the important receptors of Bt toxin, which is closely related to the insecticidal mechanism of Bt toxin and the production of insect Bt resistance. In this study, we cloned the cDNA sequence of Sesamia inferensBt receptor protein aminopeptidase N (APN) by degenerate primer PCR combined with RACE, and analyzed the homology alignment of NCBI. Because of the APN3 gene, it is named SiAPN3 (GenBank Accession No. HQ636624). Sequence analysis showed that the full-length cDNA sequence of this gene was 3411bp and the open reading frame was 3018bp, which encoded 1006 amino acids. The molecular weight of the predicted protein was 114kD and the isoelectric point was 4.95. The deduced amino acid sequence of Lepidoptera insect aminopeptidase A typical structural feature of enzyme N, a cleavage signal peptide containing one N- and 12 O-linked glycosylation sites with a 18 amino acid at the N-terminus, a zinc glutamine aminopeptidase conserved structure GAMEN , The zinc binding site HEXXHX18E, and a 22 amino acid glycosylphosphatidylinositol (GPI) anchor signal peptide at the C-terminus. The transcriptional profiling of SiAPN3 in different parts of the intestinal tract and larvae of 4th instar larvae of S. avenae was studied by real-time quantitative PCR. The results showed that the expression of this gene was the highest in the midgut of larvae, followed by the hindgut, the lowest in the foregut and the higher in the midgut and hindgut (P <0.05). The expression of SiAPN3 in the midgut The 3rd instar larvae had the highest expression level and the lowest larvae in the 1st instar larvae. Although there was no significant difference between the 3rd and 4th larvae, both of them were significantly higher than other days, There was no significant difference (P> 0.05). This study laid the foundation for clarifying the function of APN gene and the molecular mechanism of Stem borer resistance to Bt.