摘要
美国白蛾(Hyphantria cunea)是桑树的重要害虫。为明确苏云金杆菌(Bacillus thuringiensis)杀虫晶体蛋白对该虫的作用机制,利用已知昆虫氨肽酶N基因序列设计引物,以美国白蛾中肠cDNA为模板,利用RACE-PCR技术获得美国白蛾中肠氨肽酶N基因hcapn1全长序列(GenBank登录号:KP053647)。该基因开放阅读框为3 000 bp,编码999个氨基酸,预测蛋白质的分子质量和等电点分别为113.14 kD和4.85。设计去信号肽引物PCR扩增获得hcapn1基因序列,构建原核重组表达载体pET30a-hcapn1,诱导表达的HcAPN1重组蛋白约110 kD。将苏云金杆菌Cry1Ac、Cry2Ab33和Cry9Ea6原毒素经胰蛋白酶消化后与HcAPN1重组蛋白分别进行体外配体印迹试验,发现HcAPN1重组蛋白与Cry9Ea6结合,而不与Cry2Ab33、Cry1Ac发生特异结合,推测HcAPN1可能为Cry9Ea6在美国白蛾中肠的受体蛋白。分别设计引物扩增HcAPN1蛋白2个结构域Asp_(34)~Asp_( 527)和Leu_(563)~Gln_(925)的编码序列,在大肠埃希菌中表达获得62 kD和48 kD 2种重组蛋白,体外结合试验显示Cry9Ea6与HcAPN1的结合发生在Leu_(563)~Gln_(925)之间。研究结果为深入理解苏云金杆菌Cry9Ea6蛋白对美国白蛾的杀虫机制提供了基础数据。
Hyphantria cunea Drury is an important pest of Morus alba L.. In order to classify the action mechanism of insecticidal toxins from Bacillus thuringiensis on H. cunea,the full length cDNA sequence of H. cunea midgut aminopeptidase N gene hcapn1( GenBank accession No.KP053647) was obtained through RACE-PCR using H.cunea midgut cDNA as template and specific primers,which was designed according to known insect aminopeptidase N gene sequence. The open reading frame of hcapn1 was 3 000 bp,encoding 999 amino acid residues. The predicted molecular weight and isoelectric point of HcAPN1 were 113. 14 k D and 4. 85,respectively.Signal peptide-removed hcapn1 gene was cloned by PCR and used to construct recombinant expression vector pET30 a-hcapn1 for prokaryotic expression. The molecular weight of induced HcAPN1 recombinant protein was about 110 k D. In vitro ligand blot assay was performed to ascertain the binding activity of HcAPN1 recombinant protein with B. thuringiensis bysepticus Cry1 Ac,Cry2 Ab33 and Cry9 Ea6 toxins after treated with trypsin. Results showed that HcAPN1 recombinant protein can bind specifically to Cry9 Ea6,while could not bind to Cry1 Ac and Cry2 Ab33. It was suggested that HcAPN1 might be the receptor of Cry9 Ea6 in midgut of H.cunea. Primers were designed to amplify the coding regions of two functional domains( Asp34-Asp527 and Leu563-Gln925)of HcAPN1 protein,respectively,and two recombinant proteins with molecular weight of 62 k D and 48 k D were expressed in Escherichia coli. In vitro binding test showed that the binding of Cry9 Ea6 with HcAPN1 occurred between Leu563 and Gln925. These results provide basic data for further understanding the mechanism of Cry9 Ea6 protein on H. cunea.
引文
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