关键词: 暗黑鳃金龟, V-ATPase B, 苏云金芽孢杆菌, Cry8Ea3毒素, ELISA, 免疫荧光分析, 细胞毒性

Abstract:

【Objective】 This study aims to investigate the function of HpvATPase B protein, and to clarify the role of this protein in the action of Bacillus thuringiensis (Bt) crystal protein against the larvae of Holotrichia parallela. 【Method】 Based on transcriptome data of the H. parallela, the open reading frame (ORF) of HpvATPase B was identified and cloned. HpvATPase B was expressed in vitro using a prokaryotic expression system and detected by Western blot. The expression levels of HpvATPase B in different tissues of 2-day-old of 3rd instar larvae of H. parallela were determined using qRT-PCR. The binding characteristics of HpvATPase B protein to Bt Cry8Ea3 toxin were detected by Ligand blot and ELISA. Sf9 cells transfected with HpvATPase B were subjected to immunofluorescence and cell viability assays to evaluate the binding of HpvATPase B to Bt Cry8Ea3, and the changes in cell mortality after treatment with Cry8Ea3 were compared. 【Result】 The cloned HpvATPase B (GenBank accession number: MZ004965) is about 1 497 bp, encoding 498 amino acids with a predicted molecular weight of 55 kDa and an isoelectric point (pI) of 5.51. Three N-glycosylation sites (²³⁹N, ³³³N, ⁴⁵⁸N) and four O-glycosylation sites (⁴S, ⁸T, ²³S, ²⁸S) were predicted. HpvATPase B protein has the highest sequence identity (55%) with Trypoxylus dichotomus V-ATPase B (GenBank accession number: GJQ75664.1) and Oryctes borbonicus V-ATPase B (GenBank accession number: KRT83436.1). The recombinant plasmid pET30a-HpvATPase B was successfully constructed, yielding a 55 kDa protein with peak expression at 8 h post-induction. The HpvATPase B had the highest expression level in the Malpighian tubules. Ligand blot confirmed specific binding between HpvATPase B and Bt Cry8Ea3 but not Cry1Ab35. The affinity of HpvATPase B protein to Bt Cry8Ea3 and Cry1Ab35 was determined by ELISA. The binding ability to Bt Cry8Ea3 was strong, and the Kd was 7.20 nmol·L^-1, but it did not bind to Cry1Ab35, and the affinity did not change with the concentration of Cry1Ab35. pFastBac^TM HTA-HpvATPase B was constructed and Sf9 transgenic cells were successfully obtained. Immunofluorescence assay showed that HpvATPase B protein specifically bound to Cry8Ea3 toxin protein. Cell bioassay showed that when the concentration of Cry8Ea3 protein was 10 and 100 μg·mL^-1, the average corrected mortality of transgenic cells was 25.92% and 75.53%, respectively, and the difference was significant (P<0.05), indicating that HpvATPase B was Bt Cry8Ea3 receptor protein. 【Conclusion】 HpvATPase B protein was identified as the receptor protein of Bt Cry8Ea3 through a series of in vitro binding assays, immunofluorescence analyses, and cytotoxicity evaluations. This protein plays a crucial role in mediating the toxic effects of Bt Cry8Ea3 on H. parallela larvae.

Key words: Holotrichia parallela, V-ATPase B, Bacillus thuringiensis, Cry8Ea3 toxin, ELISA, immunofluorescence analysis, cytotoxicity