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精氨酸激酶(arginine kinase,AK)是一种磷酸转移酶,参与无脊椎动物的细胞内能量代谢。为深入了解AK在棉铃虫(Helicoverpa armigera)细胞色素P450(cytochrome P450)CYP6B6基因表达过程中起到的作用和调控机理,基于酵母单杂交结果采用RT-PCR从棉铃虫中肠c DNA扩增得到棉铃虫精氨酸激酶(Harm AK),构建大肠杆菌(Escherichia coli)重组菌株BL21:p ET28a-Harm AK,异丙基硫代半乳糖苷(isopropylβ-D-thiogalactoside,IPTG)诱导表达后通过镍柱纯化获得目的蛋白,Western blot检测目的蛋白的表达和纯化结果,p H-分光光度法检测Harm AK蛋白的催化活性,q RT-PCR检测棉铃虫不同发育阶段和6龄幼虫不同组织中Harm AK基因的表达规律。研究结果表明,克隆得到Harm AK基因大小为1 068 bp,编码355个氨基酸,预测蛋白质分子量和等电点分别是39.8 k D和5.76。氨基酸序列分析表明,Harm AK蛋白不含信号肽和跨膜结构域。Harm AK在大肠杆菌BL21中为可溶蛋白,Western blot和p H-分光光度法结果显示,融合蛋白His-Harm AK的大小与预期一致,且活性达到(5.5±0.85)μmol/(min·mg),表明Harm AK能磷酸化L-精氨酸。Harm AK在棉铃虫的所有发育阶段和6龄幼虫的所有组织中均有表达,1龄幼虫及6龄幼虫中肠的表达量最高。本研究将为利用Harm AK基因作为分子靶标来控制棉铃虫的研究奠定基础。
Arginine kinase (AK) is a phosphotransferase involved in intracellular energy metabolism in invertebrates. In order to further understand the role and regulation mechanism of AK in the CYP6B6 gene expression in Helicoverpa armigera cytochrome P450, based on the yeast single hybridization results, Helicoverpa armigera arginine kinase (Harm AK), Escherichia coli recombinant strain BL21: p ET28a-Harm AK and isopropylβ-D-thiogalactoside (IPTG) Purification was used to obtain the target protein. Western blot was used to detect the expression and purification of the target protein. The catalytic activity of Harm AK protein was detected by p H-spectrophotometry. The expression of Harm AK was detected by q RT-PCR at different developmental stages and in 6th instar larvae Gene expression patterns. The results showed that the cloned Harm AK gene was 1 068 bp, encoding 355 amino acids. The molecular weight and isoelectric point of the predicted protein were 39.8 kD and 5.76, respectively. Amino acid sequence analysis showed that Harm AK protein does not contain signal peptide and transmembrane domain. Harm AK was soluble protein in E.coli BL21. The results of Western blot and p H-spectrophotometry showed that the size of fusion protein His-Harm AK was as expected and the activity reached (5.5 ± 0.85) μmol / (min · mg ), Indicating that Harm AK phosphorylates L-arginine. Harm AK was expressed in all tissues of the 6th instar larvae at all developmental stages of H. armigera and the highest expression was observed in the midgut of the 1st instar larvae and the 6th instar larvae. This study will lay the foundation for the study of controlling the cotton bollworm by using the Harm AK gene as a molecular target.