Mutation of an aminopeptidase N gene is associated with Helicoverpa armigera resistance to Bacillus thuringiensis Cry1Ac toxin.
Mutation of an aminopeptidase N gene is associated with Helicoverpa armigera resistance to Bacillus thuringiensis Cry1Ac toxin.
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DOI：
10.1016/j.ibmb.2009.04.003
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发表时间：
2009-07
影响因子：
3.8
通讯作者：
Shaoping Zhang;Hongmei Cheng;Yulin Gao;Gui-rong Wang;G. Liang;Kongming Wu
中科院分区：
文献类型：
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作者：
Shaoping Zhang;Hongmei Cheng;Yulin Gao;Gui-rong Wang;G. Liang;Kongming Wu
用Cry1Ac毒素筛选75代，获得了一株抗性2971倍的棉铃虫抗Cry1Ac毒株Bt-R。我们使用dna扩增片段长度多态性分析鉴定了cry1ac耐药和敏感菌株中差异表达的基因，在2000个筛选的cdna中发现了212个差异表达转录本。在这些转录衍生片段（tdf）中，有37个与已知序列有一定的同源性，包括氨基肽酶N (APN)，它在中肠上皮中表达，在包括棉蚜在内的几个月中作为Cry1A亚家族受体存在。我们通过RT-PCR证实了TDF，并在Bt-R菌株中发现了apn1的缺失突变。我们在细菌中表达了TDF。部分HaAPN1-96S野生型蛋白在配体印迹上与Cry1Ac结合，而HaAPN1-BtR不与Cry1Ac结合。这表明HaAPN1是Bt Cry1Ac的受体，其缺失突变与棉铃虫的Cry1Ac抗性有关。缺乏一个结合位点是其对Cry1Ac具有抗性的原因。我们开发了一种等位基因特异性PCR来监测棉铃虫田间种群中apn1基因是否产生类似的突变。2006-2007年野外采集的2250例个体未发现缺失突变体。
A Cry1Ac-resistant strain (Bt-R) of Helicoverpa armigera, with 2971-fold resistance, was derived by selection with Cry1Ac toxin for 75 generations. We used cDNA-amplified fragment length polymorphism analysis to identify those genes differentially expressed in the Cry1Ac-resistant and -susceptible strains, which revealed 212 differentially expressed transcripts among 2000 screened cDNAs. Among these transcript-derived fragments (TDFs), 37 showed some homology to known sequences, including Aminopeptidase N (APN), which is expressed in the midgut epithelium and has been implicated as a Cry1A subfamily receptor in several moths, including H. armigera. We confirmed the TDF by RT-PCR and identified a deletion mutation of apn1 in the Bt-R strain. We expressed the TDF in bacteria. The partial HaAPN1-96S wild-type protein, bound to Cry1Ac on ligand blots, whereas HaAPN1-BtR did not. This suggested that HaAPN1 is a receptor for Bt Cry1Ac and that its deletion mutation is associated with Cry1Ac resistance in H. armigera. The absence of one binding site is responsible for its resistance to Cry1Ac. We developed an allele-specific PCR to monitor whether the apn1 gene in an H. armigera field population produced a similar mutation. No deleted mutants were found in 2250 individuals collected from the field in 2006–2007.