Scientia Agricultura Sinica ›› 2025, Vol. 58 ›› Issue (10): 1947-1957.doi: 10.3864/j.issn.0578-1752.2025.10.007

• PLANT PROTECTION • Previous Articles     Next Articles

Cloning，Expression and Binding Characteristic with Bt Cry8Ea3 Toxin of HpvATPase B from Holotrichia parallela

QIAO YingCui1(), WANG BoYu1(), WANG Qian1, ZHAO Dan1(), GUO Wei2, NING WenShuo3, CHANG MengYing4, WANG Hai1, LU XiuJun1()   

  1. 1 College of Plant Protection, Hebei Agricultural University, Baoding 071001, Hebei
    2 Graduate School of Chinese Academy of Agricultural Sciences, Beijing 100081
    3 Ningjin County Agriculture and Rural Bureau, Ningjin 055550, Hebei
    4 Zhuozhou County Agriculture and Rural Bureau, Zhuozhou 072750, Hebei
  • Received:2025-01-17 Accepted:2025-03-24 Online:2025-05-16 Published:2025-05-21
  • Contact: ZHAO Dan, LU XiuJun

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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.

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

Table 1

The primer sequences used in this study"

引物名称 Primers name 引物序列 Primer sequence (5′-3′) 作用 Function
HpvATPase B-F1 CG GGATCCGCTGTGTTGAACTGTTGTAT 原核表达
Prokaryotic expression
HpvATPase B-R CCC AAGCTTGGGATAGAATTCTGCCAT
HpvATPase B-F2 GGATCCGGCTGTGTTGAACTGTTGTAT 真核表达
Eukaryotic expression
HpvATPase B-R CCC AAGCTTGGGATAGAATTCTGCCAT
qvATPase B-F TGGCGTAAATGGACCTCT 组织表达
Tissue expression
qvATPase B-R CAATACCCGATGTTCCCTC
Actin-F ATGTTGCCATCCAAGCTGTA
Actin-R CCAAACGCAAAATAGCATGA
M13-F CGCCAGGGTTTTCCCAGTCACGAC 重组Sf9细胞黏粒检测
Detection of recombinant cosmid in Sf9 cells
M13-R CACACAGGAAACAGCTATGAC

Fig. 1

Phylogenetic tree of V-ATPase B amino acid sequences of H. parallela and other Coleoptera species"

Fig. 2

Verification of pET30a-HpvATPase B and detection of prokaryotic expression products of HpvATPase B protein A：重组表达载体pET30a-HpvATPase B酶切验证Verification of recombinant vector pET30a-HpvATPase B。M：λ-EcoT14 I digest Marker；1：HpvATPase B；2：pET30a；3：pET30a-HpvATPase B/BamH I＆Hind III；4：pET30a-HpvATPase B/Hind III。B：重组表达载体pET30a-HpvATPase B在大肠杆菌BL21（DE3）中的表达Expression of recombinant expression vector pET30a-HpvATPase B in a bacterial system E. coli BL21 (DE3)。M：蛋白Marker Protein Marker；1：未经IPTG诱导Not induced by IPTG；2—4：HpvATPase B分别经IPTG诱导4、6、8 h HpvATPase B was induced by IPTG for 4, 6, 8 h, respectively。C：重组蛋白HpvATPase B的纯化Purification of recombinant protein HpvATPase B。M：预染Marker Prestained Marker；1：未经IPTG诱导Not induced by IPTG；2—4：HpvATPase B分别经IPTG诱导4、6、8 h HpvATPase B was induced by IPTG for 4, 6, 8 h, respectively"

Fig. 3

Expression levels of HpvATPase B in different tissues of H. parallela The different lowercases on the bars indicate that the analysis of variance is significantly different at the 0.05 level"

Fig. 4

Ligand blot analysis of Bt Cry8Ea3 and HpvATPase B protein A：Cry8Ea3活性蛋白的检测Detection of Cry8Ea3 active protein。M：蛋白Marker Protein Marker；1：Cry8Ea3原毒素Cry8Ea3 protoxin；2：Cry8Ea3活性蛋白Cry8Ea3 active protein。B：BBMV与Cry8Ea3的Ligand blot分析Ligand blot analysis of BBMV and Cry8Ea3。M：预染Marker Prestained Marker；1：BBMV与Cry8Ea3的Ligand blot分析Ligand blot analysis of BBMV and Cry8Ea3。红色箭头表示结合范围大小，依次为70、40—60、35 kDa The red arrow indicates the size of the combination range, which is about 70, 40-60 and 35 kDa；2：BBMV未与Cry8Ea3孵育BBMV not incubated with Cry8Ea3。C：HpvATPase B与Cry8Ea3、Cry1Ab35的Ligand blot分析Ligand blot analysis of HpvATPase B and Cry8Ea3, Cry1Ab35。M：预染Marker Prestained Marker；1：HpvATPase B与Cry8Ea3孵育Incubate HpvATPase B with Cry8Ea3；2：HpvATPase B未与Cry1Ab35孵育HpvATPase B not incubated with Cry1Ab35"

Fig. 5

Determination of affinity between HpvATPase B and Cry8Ea3"

Fig. 6

Identification of recombinant vector pFastBacTM HTA-HpvATPase B by enzyme digestion and the expression of HpvATPase B protein in Sf9 cells A：重组载体pFastBacTM HTA-HpvATPase B酶切Enzyme digestion of recombinant vector pFastBacTM HTA-HpvATPase B。M：λ-EcoT14 I digest Marker；1：pFastBacTM HTA；2：HpvATPase B PCR产物PCR product of HpvATPase B；3：pFastBacTM HTA-HpvATPaseB/BamH I＆Hind III；4：pFastBacTM HTA-HpvATPase B/Hind III。B：重组Bacmid PCR鉴定PCR identification of recombinant Bacmid。M：DL5000；1：pFastBacTM HTA-HpvATPase B。C：P2代病毒电泳检测Electrophoresis detection of P2。M：DL5000 Marker；1：pFastBacTM HTA对照扩增产物pFastBacTM HTA control amplification product；2：pFastBacTM HTA-HpvATPase B扩增产物pFastBacTM HTA-HpvATPase B amplification product。D：HpvATPase B蛋白在Sf9细胞的表达Expression of HpvATPase B protein in Sf9 cells。M：预染Marker Prestained Marker；1：P3对照（仅含pFastBacTM HTA）P3 control (pFastBacTM HTA only)；2：P3转基因细胞（含pFastBacTM HTA-HpvATPase-B）P3 transgenic cells (containing pFastBacTM HTA-HpvATPase-B)"

Fig. 7

Combination of HpvATPase B to Cry8Ea3 toxin protein in Sf9 cells"

Fig. 8

Cytotoxicity of different concentrations of Cry8Ea3 toxin protein to trans-HpvATPase B Sf9 cells"

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