中国农业科学2025，Vol.58Issue(10)：1947-1957,11.DOI:10.3864/j.issn.0578-1752.2025.10.007
暗黑鳃金龟HpvATPase B克隆、表达及与Bt Cry8Ea3毒素结合特性
Cloning,Expression and Binding Characteristic with Bt Cry8Ea3 Toxin of HpvATPase B from Holotrichia parallela
摘要
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.pFastBacTM 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.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Key words
Holotrichia parallela/V-ATPase B/Bacillus thuringiensis/Cry8Ea3 toxin/ELISA/immunofluorescence analysis/cytotoxicity引用本文复制引用
乔英翠,王薄毓,王倩,赵丹,郭巍,宁文烁,常梦颖,王海,陆秀君..暗黑鳃金龟HpvATPase B克隆、表达及与Bt Cry8Ea3毒素结合特性[J].中国农业科学,2025,58(10):1947-1957,11.基金项目
国家现代农业产业技术体系(CARS-13)、河北省中央引导地方项目(246Z6507G)、石家庄市驻冀高校重点研发专项(241490102A)、河北省现代农业产业技术体系(HBCT2024190208,HBCT2024130204) （CARS-13）
