RESUMEN

Several receptor proteins of Cry toxin have been previously identified, including cadherin-like, aminopeptidase N, and alkaline phosphatase. In the present work, a novel binding protein, V-ATPase subunit A (HpVAA), was identified in Holotricia parallela larvae and characterized. We performed reverse transcription-polymerase chain reaction and rapid amplification of cDNA ends technology to obtain the cDNA of the full-length hpvaa. Sequencing analysis showed that the open reading frame of hpvaa (GenBank accession No. KU497557) is 1845 bp long, encoding 614 amino acid residues. The predicted molecular weight and isoelectric point of HpVAA were 67.85 kDa and 4.9, respectively. The HpVAA protein, which includes 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, possesses the same structural characteristics as V-ATPase subunit A from other insects. The protein was successfully expressed in Escherichia coli, and a ligand blot assay showed binding of the protein with Cry8Ea3 toxin. Transcriptional analysis of hpvaa in different tissues of H. parallela larvae was performed by qRT-PCR, which showed that the relative expression of hpvaa in the Malpighian tubules is higher than that in other tissues.

Asunto(s)

Proteínas Bacterianas/química , Escarabajos/genética , Endotoxinas/química , Proteínas Hemolisinas/química , Proteínas de Insectos/química , Filogenia , Subunidades de Proteína/química , ATPasas de Translocación de Protón Vacuolares/química , Secuencia de Aminoácidos , Animales , Toxinas de Bacillus thuringiensis , Proteínas Bacterianas/genética , Secuencia de Bases , Clonación Molecular , Escarabajos/clasificación , Escarabajos/inmunología , Escarabajos/microbiología , Secuencia Conservada , ADN Complementario/genética , ADN Complementario/metabolismo , Endotoxinas/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Expresión Génica , Proteínas Hemolisinas/genética , Proteínas de Insectos/genética , Proteínas de Insectos/inmunología , Punto Isoeléctrico , Larva/genética , Larva/microbiología , Túbulos de Malpighi/inmunología , Túbulos de Malpighi/metabolismo , Túbulos de Malpighi/microbiología , Modelos Moleculares , Peso Molecular , Sistemas de Lectura Abierta , Unión Proteica , Subunidades de Proteína/genética , Subunidades de Proteína/inmunología , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/inmunología , ATPasas de Translocación de Protón Vacuolares/genética , ATPasas de Translocación de Protón Vacuolares/inmunología