Scientia Agricultura Sinica ›› 2019, Vol. 52 ›› Issue (19): 3337-3345.doi: 10.3864/j.issn.0578-1752.2019.19.005

• PLANT PROTECTION • Previous Articles     Next Articles

Effect of Midgut Specific Binding Protein ABCC1 on Cry1Ac Toxicity Against Helicoverpa armigera

CHEN Lin1,WEI JiZhen2,LIU Chen1,NIU LinLin1,ZHANG CaiHong1,LIANG GeMei1()   

  1. 1 State Key Laboratory for Biology of Plant Diseases and Insect Pests, Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing 100193
    2 College of Plant Protection, Henan Agricultural University, Zhengzhou 450002
  • Received:2019-04-23 Accepted:2019-05-27 Online:2019-10-01 Published:2019-10-11
  • Contact: GeMei LIANG E-mail:gmliang@ippcaas.cn

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Abstract:

【Objective】 The objective of this study is to research the binding characteristics of midgut protein ABCC1 from Helicoverpa armigera (HaABCC1) to Cry1Ac and the effect of ABCC1 on toxicity of Cry1Ac, and to clarify the role of HaABCC1 in the insecticidal mechanism of Cry1Ac. 【Method】 To verify the binding ability of HaABCC1 with Cry1Ac in vitro, the sequence of HaABCC1 was analyzed and the primers were designed, two heterologously transmembrane protein fragments of HaABCC1 were expressed, and then Ligand blot experiment was conducted. The changes of expression level of HaABCC1 and mortality of larvae exposed to Cry1Ac were tested after silenced HaABCC1 by microinjected siRNA into the abdomen of 3rd instar larvae. The cell mortality changes of Sf9 insect cell line treated by Cry1Ac were compared using cell bioassay method, after transfected HaABCC1 into Sf9 and confirmed the pAc-ABCC1 recombinant plasmid was successfully transferred into Sf9. The difference of full-length HaABCC1 between Cry1Ac-resistant strain (BtR) and susceptible strain (96S) was compared, the expression of HaABCC1 in BtR and 96S was detected using qRT-PCR. 【Result】 The two HaABCC1 transmembrane fragments, TMD1 and TMD2, were successfully expressed in Escherichia coli BL21 (DE3) cells, these two heterologously expressed HaABCC1 fragments could bind to actived-Cry1Ac in vitro. The expression level of HaABCC1 significantly reduced after injection of siABCC1 into abdomen of the H. armigera larvae. Compared with non-treated, DEPC water- and siEGFP-injected larvae, the mortality of H. armigera larvae which were knocked down HaABCC1 significantly decreased after treated with actived-Cry1Ac. It was showed the toxicity of Cry1Ac against H. armigera larvae significantly reduced after the HaABCC1 silence. Compared with Sf9 control, the mortality of cell line which HaABCC1 expressed in Sf9 obviously increased after treated by actived-Cry1Ac. The result indicated that transfection HaABCC1 into Sf9 could significantly increase the cell mortality after Cry1Ac treatment. There was no difference in the cDNA sequence and amino acid sequence of HaABCC1 between susceptible strain (96S) and Cry1Ac-resistant strain (BtR), but the expression level of HaABCC1 significantly reduced in BtR resistant strain compared with 96S susceptible strain. 【Conclusion】 HaABCC1 of H. armigera is a specific binding protein and may be a functional receptor protein of Cry1Ac, it may involve in the resistance of H. armigera to Cry1Ac.

Key words: Helicoverpa armigera, ABCC1, Bacillus thuringiensis, Cry1Ac, binding protein, toxicity

Table 1

Listing of primers used in RT-PCR, qRT-PCR and RNAi reactions"

引物名称
Primer name		 引物序列
Primer sequence (5′- 3′)
ABCC1-F TTAATTAACCAGTCCGGCGTCC
ABCC1-R ATGTCTTACAATTCTACGCTTGA
GAPDH-F CATTGAAGGTCTGATGACCACTGT
GAPDH-R CAGAGGGTCCATCCACTGTCTT
GAPDH-Probe CACGCCACCATTGCCACCCA
β-actin-F GGCCCCGTCCACAATGA
β-actin-R CCGATCCATACGGAGTACTTCCT
β-actin-Probe ATCAAGATCATCGCGCCCCCAGA
ABCC1-F GGCAGCGTGAAAAGAAAGAC
ABCC1-R GTGATCACGGACGAGAGGAT
ABCC1-Probe CGAAGCTGAGAAAACCGAGACTGGAAG
TMD1-EcoRV-F GATATCGCGTTCGGCGGTCAGTTC
TMD1-HindIII-R AAGCTTACGTGACATCGAGTTCATCACAATT
TMD2-EcoRV-F GATATCTTGATGAGCGTCGGAATCCT
TMD2-HindIII-R AAGCTTATGTTTCTACTTCTGATGTCATGCGTAC
siRNA-ABCC1 GGAUGUACCUGGUGGGCAUTT
siRNA-EGFP GCGUUGGGAAGUCAAGUUUTT
ABCC1-PmeI-F GTTTAAACATGTCTTACAATTCTACGCTTG
ABCC1-StuI-R AGGCCTTTAATTAACCAGTCCGGCGTCC
ABCC1-F’ AGAAGCCTGCCTCCATACTACC
ABCC1-R’ AAGAAGCGCAACTGCATAAAC

Fig. 1

SDS-PAGE of HaABCC1 fragments by prokaryotic expression and Ligand blot detection of Cry1Ac binding with HaABCC1"

Fig. 2

HaABCC1 expression after gene silencing (A) and its effect on H. armigera susceptibility to Cry1Ac (B) Different lowercases indicate significant differences among treatments (P＜0.05). The same as below"

Fig. 3

Effects of pAc-ABCC1 plasmid transfection into Sf9 cells on the mortality after treated with activated Cry1Ac"

Fig. 4

The expression level of HaABCC1 in different strains of H. armigera"

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