亚洲玉米螟中肠Cry1Ac毒素结合蛋白V-ATP酶A亚基的研究

Cloning and Characterization of the Cry1Ac-binding V-ATPase Subunit A in the Asian Corn Borer

运用RT-PCR和RACE技术克隆了亚洲玉米螟Ostrinia furnacalis(Guenée)幼虫中肠V-ATP酶A亚基基因ofvaa,其c DNA全序列长2535 bp,开放阅读框1854 bp,编码617个氨基酸,预测蛋白分子量68.01 k D,等电点(p I)4.90。序列中包含有V-ATP酶A亚基特征,即ATP-synt_ab_N结构域ATP-synt_ab_C结构域和可供ATP结合的保守区Walker A(GAFGCGKT),以及天门冬氨酸介导Mg2+反应的保守区Walker B(SMMAD)。原核表达并获得纯化的Of VAA多肽片段,配体杂交试验表明其能够与Cry1Ac毒素结合。生测结果表明,Of VAA多肽片段对亚洲玉米螟有一定的杀虫活性,当其与Cry1Ac毒素混合时杀虫效果呈现加性效应。鉴定的亚洲玉米螟V-ATP酶A亚基基因ofvaa c DNA序列在Gen Bank登录号为HQ434762。

Using RT-PCR and RACE, the full-length cDNA （2535 bp） coding a V-ATPase subunit A （VAA） gene ofvaa was cloned and sequenced from the larval midguts of the Asian corn borer Ostriniafurnacalis （Guen6e）. The relative open reading frame （ORF） sequence consisted of 1854 nucleotides, which translated into 617 amino acids, coding for the OfVAA with predicted molecular weight of 68.01 kD and isoelectric point （pI） of 4.90. The sequence was identified as VAA because it shared the common structural features with V-ATPase subunit A, including two putative conserved domains ATP-synt_ab_N and ATP-synt ab_C, and a Walker A （GAFGCGKT） motif and a Walker B （SMMAD） motif. The protein was successfully expressed using Escherichia coli and purified. The ligand blot assay showed that the purified OfVAA peptide could bind with Cry 1 Ac toxin and enhance Cry 1 Ac insecticidal activity against the Asian corn borer larvae. The sequence reported in this paper has been deposited in the GenBank database at accession No.： HQ434762.