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为研究亚洲玉米螟Ostrinia furnacalis(Guenée)幼虫体内酚氧化酶原激活蛋白酶(prophenoloxidase activating proteinase,PAP)表达调控的分子机理,本研究根据不同昆虫酚氧化酶原激活蛋白酶基因序列的保守区域,设计合成简并引物,采用RT-PCR技术从亚洲玉米螟5龄幼虫中扩增出PAP的一段cDNA片段,大小为509bp,编码169个氨基酸,预测分子量为18.7kD,理论等电点(pI值)为5.1。该基因序列中含有丝氨酸蛋白酶样结构域中保守的催化三联体,不含发夹结构域。BlastP分析结果表明:该片段的氨基酸序列与烟草天蛾Manduca sextaPAP-3和冈比亚按蚊Anopheles gambiae发夹型蛋白B1的氨基酸序列一致性最高,为47%;与烟草天蛾PAP-2、家蚕BombyxmoriPPAE、斜纹夜蛾Spodoptera lituraPPAE-3、蓖麻蚕Samia cynthia riciniPAP和黑腹果蝇Drosophila melanogaster丝氨酸蛋白酶7的氨基酸序列的一致性分别为45%,45%,44%,43%和41%。构建系统发育树,对其进化关系进行了初步分析,结果显示:亚洲玉米螟PAP与烟草天蛾PAP-3和斜纹夜蛾PPAE-3的亲缘关系较近,与黑腹果蝇丝氨酸蛋白酶7和冈比亚按蚊发夹型蛋白B1的亲缘关系较远。这些结果说明克隆得到的cDNA片段为亚洲玉米螟幼虫PAP基因靠近羧基端的部分序列。
In order to study the molecular mechanism of the expression of prophenoloxidase activating proteinase (PAP) in the larvae of Ostrinia furnacalis (Guenée), we designed and synthesized the conserved regions of different PRS gene sequences Degenerate primers were used to amplify a cDNA fragment of PAP from the 5th instar larvae of Asian corn borer, RT-PCR, with a size of 509bp and encoding a polypeptide of 169 amino acids with a predicted molecular weight of 18.7 kD. The theoretical isoelectric point 5.1. The gene sequence contains a conserved catalytic triad of the serine protease-like domain without hairpin domains. The results of BlastP analysis showed that the amino acid sequence of the fragment was 47% identical to the amino acid sequence of Manduca sextaPAP-3 and Anopheles gambiae hairpin protein B1, and the amino acid sequence of the fragment was the same as that of PAP-2, Bombyx mori PPAE , 45%, 45%, 44%, 43% and 41% identity with Spodoptera lituraPPAE-3, Samia cynthia riciniPAP and Drosophila melanogaster serine proteinase 7, respectively. The phylogenetic tree was constructed and the phylogenetic relationship was analyzed. The results showed that PAP of Asian corn borers was closer to that of PAP-3 and PPAE-3, but not to that of sericin proteinase 7 and Gambia Anopheline hairpin protein B1 is more distantly related. These results indicate that the cloned cDNA fragment is a partial sequence close to the carboxyl terminal of the PAP gene of Asiatic corn borer larvae.