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为探究甜菜夜蛾Spodoptera exigua中肠碱性磷酸酶(alkaline phosphatase protein 2,ALP2)是否为Cry1Ac杀虫蛋白的受体,采用同源克隆和RACE技术克隆了编码alp2基因的完整c DNA序列,利用荧光定量PCR比较了甜菜夜蛾幼虫中肠不同龄期ALP2表达量的差异,利用Ligand blot方法检测了中肠ALP2与Cry1Ac杀虫蛋白的结合。结果表明,alp2基因序列全长1 629 bp(Gen Bank序列号为KP420013),编码542个氨基酸,预测在氨基酸序列N端包含1个由21个氨基酸组成的信号肽,在C端存在1个GPI修饰的锚定位点,且在整个氨基酸序列中存在多个糖基化修饰位点。在整个甜菜夜蛾幼虫期均有ALP2表达,但不同龄期的表达量差异显著,1龄幼虫期表达量最低,4龄幼虫期最高。Ligand blot方法检测结果表明原核表达的ALP2片段与活化的Cry1Ac杀虫蛋白可以结合。研究表明,甜菜夜蛾中肠的ALP2可能是Cry1Ac的受体之一。
To investigate whether alkaline phosphatase protein 2 (ALP2) of Spodoptera exigua is a receptor of Cry1Ac insecticidal protein, the complete cDNA sequence of alp2 gene was cloned by homologous cloning and RACE technique. Fluorescence quantitative PCR was used to compare the expression of ALP2 in different stages of midgut of beet armyworm larvae, and the combination of ALP2 and Cry1Ac insecticidal protein in midgut was detected by Ligand blot. The results showed that the full length of alp2 gene was 1 629 bp (GenBank accession number KP420013) encoding 542 amino acids. The predicted signal sequence of alp2 gene consisted of a signal peptide consisting of 21 amino acids at the N terminus and a GPI at the C terminus Modified anchor sites, and multiple glycosylation sites exist throughout the amino acid sequence. ALP2 expression was observed throughout the larvae of beet armyworm, but the expression level was significantly different at different ages, with the lowest expression in the first instar larvae and the highest in the fourth instar larvae. The results of Ligand blot showed that the prokaryotic expressed ALP2 fragment could bind with the activated Cry1Ac insecticidal protein. Studies have shown that ALP2 in the midgut of beet armyworm may be one of the receptors for Cry1Ac.