甜菜夜蛾中肠碱性磷酸酶alp2基因的克隆、表达及功能分析

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。

Cloning, expression and functional analysis of alkaline phosphatase 2 (alp2) in the midgut of beet armyworm Spodoptera exigua (Hübner) (Lepidoptera:Noctuidae)

To clarify the alkaline phosphatase (ALP2), the receptor of Cry1Ac, the full-length cDNA encoding ALP2 protein was cloned from the midgut of beet armyworm Spodoptera exigua with the homology cloning and RACE methods, and the binding of ALP2 with Cry1Ac was tested by ligand blot analysis. The expression levels of ALP2 in different larval developmental periods were compared by using real-time quantitative PCR. The results showed that the length of alp2 gene was 1 629 bp (GenBank accession no. KP420013) and encoded 542 amino acid residues, and the amino acid sequence contained a signal peptide of 21 amino acid residues in the N-terminus region and a GPI anchor site in the C-terminus region, and contained a few potential glycosylation sites. The ALP2 could be expressed in the whole larval period, and there were significant differences among different instars. The lowest and highest expression levels appeared in the 1st and 4th instar larvae. Ligand blot analysis results showed that recombinant ALP2 could bind with Cry1Ac insecticidal protein. The results indicated that ALP2 might be one of the receptor proteins of Cry1Ac insecticidal protein in S. exigua.