棉铃虫中肠特异性结合蛋白ABCC1对Cry1Ac毒力的影响

doi:10.3864/j.issn.0578-1752.2019.19.005

摘要/Abstract

摘要：

【目的】 研究棉铃虫（Helicoverpa armigera）中肠蛋白ABCC1（HaABCC1）与Cry1Ac的结合特性及对Cry1Ac毒力的影响,明确HaABCC1在Cry1Ac杀虫机制中的作用。【方法】 分析HaABCC1基因序列,设计引物,通过原核表达得到HaABCC1两个跨膜区片段的蛋白,与Cry1Ac进行Ligand blot试验,验证其与Cry1Ac的体外结合特性;利用RNAi技术干扰棉铃虫幼虫的HaABCC1,在3龄幼虫腹部注射siABCC1,比较HaABCC1的表达量及Cry1Ac处理后棉铃虫死亡率的变化;通过细胞转染将ABCC1导入Sf9细胞系中,确定pAc-ABCC1重组质粒转入Sf9细胞后,用细胞生物测定的方法比较Cry1Ac处理后细胞死亡率的变化;比较敏感品系（96S）和Cry1Ac抗性品系（BtR）棉铃虫的HaABCC1基因全长序列,并通过荧光定量RT-PCR检测HaABCC1在抗、感棉铃虫中的表达量。【结果】 HaABCC1跨膜区TMD1和TMD2在Escherichia coli BL21（DE3）感受态细胞中成功表达,两个HaABCC1跨膜区片段蛋白均能与活化的Cry1Ac在体外结合;棉铃虫注射siABCC1后,HaABCC1的表达量显著下降,与未注射的棉铃虫、注射DEPC水和siEGFP的棉铃虫相比,用活化的Cry1Ac蛋白处理HaABCC1被干扰的棉铃虫,其幼虫死亡率显著降低,表明棉铃虫幼虫的HaABCC1被干扰后,能显著降低Cry1Ac对棉铃虫的毒力;用活化的Cry1Ac蛋白处理成功转入HaABCC1的Sf9细胞,与对照Sf9细胞相比,细胞的死亡率明显上升,表明将HaABCC1导入Sf9后能显著提高Cry1Ac处理后的细胞死亡率;抗性品系（BtR）与敏感品系（96S）棉铃虫的HaABCC1氨基酸序列没有差别,但抗性品系BtR棉铃虫HaABCC1的表达量显著降低。【结论】 HaABCC1是Cry1Ac的特异性结合蛋白,可能是Cry1Ac的功能受体蛋白,并可能参与对Cry1Ac的抗性机制。

关键词: 棉铃虫, ABCC1, 苏云金芽孢杆菌, Cry1Ac, 结合蛋白, 毒力

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

陈琳,魏纪珍,刘臣,牛琳琳,张彩虹,梁革梅. 棉铃虫中肠特异性结合蛋白ABCC1对Cry1Ac毒力的影响[J]. 中国农业科学, 2019, 52(19): 3337-3345.

CHEN Lin,WEI JiZhen,LIU Chen,NIU LinLin,ZHANG CaiHong,LIANG GeMei. Effect of Midgut Specific Binding Protein ABCC1 on Cry1Ac Toxicity Against Helicoverpa armigera[J]. Scientia Agricultura Sinica, 2019, 52(19): 3337-3345.

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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