亚洲玉米螟幼虫体内酚氧化酶原激活蛋白酶cDNA片段的克隆与序列分析

Cloning and sequence analysis of a cDNA fragment of prophenoloxidase activating proteinase from larvae of the Asian corn borer,Ostrinia furnacalis Guenée(Lepidoptera:Pyralidae)

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 explore the molecular mechanism of expression and regulation of prophenoloxidase activating proteinase （PAP） in larvae of Ostrinia furnacalis （Guenée）,the sequences of conserved regions of PAP genes in different insects were used to design the degenerate primers,and a cDNA fragment with the size of 509 bp encoding 169 amino acids was amplified from the 5th larvae of O. furnacalis by RT-PCR. The fragment has a predicted molecular weight of 18.7 kD and pI value of 5.1. A conserved catalytic triad （H,D,S） in the serine protease like domain was found,but the clip domain did not exist. BlastP analysis showed that the amino acid sequence of the cloned fragment from O. furnacalis had the highest identity （47%） to those of Manduca sexta PAP-3 and Anopheles gambiae Clip B1 protein,and 45%,45%,44%,43% and 41% identity to those of M. sexta PAP-2,Bombyx mori PPAE,Spodoptera litura PPAE-3,Samia cynthia ricini PAP and Drosophila melanogaster serine protease-7,respectively. Phylogenetic analysis showed that the genetic relationship of O. furnacalis,M. sexta PAP-3 and S. litura PPAE-3 was closer,while that of O. furnacalis,Drosophila melanogaster serine protease-7 and Anopheles gambiae Clip B1 protein was more distant. The results suggest that the cloned cDNA fragment of O. furnacalis larvae is a part of the PAP gene nucleotide sequence at the C-terminus.