甜菜夜蛾中肠碱性磷酸酶alp2基因的克

发布时间：2018-02-15 21:08

本文关键词： 甜菜夜蛾 Bt杀虫蛋白碱性磷酸酶受体蛋白　出处：《植物保护学报》2017年01期 　论文类型：期刊论文

【摘要】：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。
[Abstract]:In order to investigate whether the midgut alkaline phosphatase (ALP2) of Spodoptera exigua Spodoptera exigua is the receptor of Cry1Ac insecticidal protein, the complete c DNA sequence encoding alp2 gene was cloned by homologous cloning and RACE techniques. The difference of ALP2 expression in midgut of Spodoptera exigua larvae was compared by fluorescence quantitative PCR. The binding of midgut ALP2 to Cry1Ac insecticidal protein was detected by Ligand blot method. The results showed that the sequence number of 1 629 bp(Gen Bank was KP42001313, encoding 542 amino acids. It was predicted that there was a signal peptide consisting of 21 amino acids at the N end of amino acid sequence. There was a GPI modified anchoring site in C terminal, multiple glycosylation sites in the whole amino acid sequence, and ALP2 expression in the whole larval stage of Spodoptera exigua. However, there were significant differences in the expression levels among different ages. The lowest expression level in the 1st instar larvae and the highest expression in the 4th instar stage. The results of detection by the method of. Ligand blot showed that the ALP2 fragments expressed in prokaryotic cells could bind to the activated Cry1Ac insecticidal protein. ALP2 in the midgut of Spodoptera exigua may be one of the receptors for Cry1Ac.
【作者单位】：中国农业科学院植物保护研究所植物病虫害生物学国家重点实验室;
【基金】：国家转基因生物新品种培育重大专项(2014ZX08011-002)
【分类号】：S433.4

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