关键词: 暗黑鳃金龟, 碱性磷酸酶, Bt Cry8Ea, 配体印记, ELISA

Abstract: 【Aim】 To ascertain the insecticidal mechanism of Bacillus thuringiensis (Bt) crystal protein against the larvae of the dark black chafer, Holotrichia parallela. 【Methods】 The full-length cDNA sequence of alkaline phosphatase gene HpALP of H. parallela was cloned by PCR based on the transcriptome data of H. parallela. HpALP was expressed in vitro by prokaryotic expression system and detected by Western blot. The expression levels of HpALP in different tissues (foregut, midgut, rectum, ileum, Malpighian tubules, fat body and integument) of the day-2 3rd instar larvae of H. parallela were detected using qRT-PCR. The in vitro binding characteristics of HpALP with the Bt crystal protein Cry8Ea3 was analyzed by ligand blot and ELISA. 【Results】 The full-length cDNA of HpALP (GenBank accession no.: MZ004964) of H. parallela was obtained. It is 1 536 bp in length, encoding 512 amino acids with the predicted molecular weight of 57.32 kD and the pI of 4.70. HpALP has the highest amino acid sequence identity with ALP of Onthophagus taurus. The recombinant HpALP was obtained by prokaryotic expression, and it had the highest expression level at 8 h after IPTG induction detected by Western blot. Tissue expression profiles revealed that HpALP showed the highest abundance in the larval foregut analyzed by qRT-PCR. The recombinant HpALP could specifically bind to Cry8Ea3 with the dissociation constant Kd value of 76.21±26.44 nmol/L and the maximum affinity B_max value of 0.59±0.068, but could not bind to the control Cry1Ab35 with the Kd value of 142.50±137.30 nmol/L and the B_max value of 0.013±0.005. 【Conclusion】 Based on the isolation and identification of HpALP with high affinity to Bt Cry8Ea3, we inferred that HpALP might be the receptor protein of Cry8Ea3. The results provide a theoretic basis for clarifying the action mechanism of Cry8Ea3 protein on H. parallela.

Key words: Holotrichia parallela, alkaline phosphatase, Bt Cry8Ea3, ligand blot, ELISA