Characterization of a novel Cry8Ea3-binding V-ATPase Subunit A in Holotrichia parallela.

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.