CrylAb敏感和抗性亚洲玉米螟氨肽酶N基因的克隆及序列差异分析

摘要

It is meaningful for resistance management to study the resistance mechanism of Asian com borer (Ostriniafurnacalis) to CrylAb.cDNA of aminopeptidase N (APN) from CrylAb toxin susceptible and resistant strains of Asian corn borer was cloned and sequenced.Ofapn3, a member of aminopeptidase N families, was identified.It contained 3 591 nucleotides with a 3 045 bp open reading frame that encoded 1 014 amino acids.Putative protein sequences included 8 and 4 potential N- and O-linked glycosylation sites and a zinc metal binding site motif of HEXXHX18E, which was 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.The sequences had a cleavable N-terminal signal peptide with 18 amino acids and a glycosylphosphatidylinositol (GPI) anchor signal peptide with 22 amino acids in C-terminal, which were also typical characters of lepidopteran APN proteins.Sequence analysis indicated that the deduced protein sequences were most similar to Onapn3 (GenBank accession No.ABL01483) from Ostrinia nubilalis with 96.6％ sequence identity.The predicted protein molecular weights and isoelectric points were 115.1 kD and 4.44, respectively.Forty nucleotide differences were observed in the open reading frames which translated into 10 amino acid differences in the putati,ve protein sequences.The sequences reported in this paper have been deposited in the GenBank database (accession No.EF538427and EU137839 for Ofapn3 from Cryl Ab-resistant strain ACB-AbR and susceptible strain ACB-BtS).%研究亚洲玉米螟(Ostrinia furnacalis)对CrylAb产生抗性的分子机理,对今后制定和实施合理的抗性治理策略具有重要的意义.本研究通过PCR方法克隆和测序,鉴定了CrylAb敏感-抗性亚洲玉米螟幼虫中肠氨肤酶N(APN)基因家族的一个成员Ofapn3,其cDNA全序列长3591bp,开放阅读框包括3 045 by,编码1 014个氨基酸.预测蛋白质分子量为115.1 kD,等电点(PI)为4.44.其推导的氨基酸序列中具有鳞翅目昆虫氨肽酶典型结构特征,即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酞肌醇(GPI)锚信号肤.系统分类归为第3支系.与欧洲玉米螟(Ostrinia nub ilalis)的Onapn3(GenBank登录号:ABL01483)同源性为96.6%.与CrylAb敏感亚洲玉米螟cDNA相比,抗性品系的开放阅读框中有40个碱基发生了点突变,导致氨基酸序列中有10个氨基酸改变.其中Ser735在抗性品系中突变为Pro的现象在抗性棉铃虫APN3的氨基酸突变中也被检测到.鉴定的Cryl Ab敏感和抗性亚洲玉米螟品系的Ofapn3 cDNA序列,已提交GenBank,登录号分别是EF538427和EU137839.