Holotrichia oblita Midgut Proteins That Bind to Bacillus thuringiensis Cry8-Like Toxin and Assembly of the H. oblita Midgut Tissue Transcriptome

The strain HBF-18 (CGMCC 2070), containing two genes ( -like and ), is toxic to larvae. Both Cry8-like and Cry8Ga proteins are active against this insect pest, and Cry8-like is more toxic. To analyze the characteristics of the binding of Cry8-like and Cry8Ga proteins to brush border membrane vesicle...

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Bibliographic Details
Published in:Applied and environmental microbiology 2017-06, Vol.83 (12)
Main Authors: Jiang, Jian, Huang, Ying, Shu, Changlong, Soberón, Mario, Bravo, Alejandra, Liu, Chunqing, Song, Fuping, Lai, Jinsheng, Zhang, Jie
Format: Article
Language:English
Subjects:
Actin
Adenosine triphosphatase
Alkaline phosphatase
Aminopeptidase
Animals
Assaying
Bacillus thuringiensis
Bacillus thuringiensis - genetics
Bacillus thuringiensis - metabolism
Bacterial Proteins - genetics
Bacterial Proteins - metabolism
Binding sites
Cadherins
Catalysis
Chromatography
Coleoptera - chemistry
Coleoptera - genetics
Coleoptera - metabolism
Coleoptera - microbiology
Endotoxins - genetics
Endotoxins - metabolism
Fluorescence
Gastrointestinal Tract - chemistry
Gastrointestinal Tract - metabolism
Gastrointestinal Tract - microbiology
Gene expression
Gene sequencing
Gram-positive bacteria
Hemolysin Proteins - genetics
Hemolysin Proteins - metabolism
Hexachaeta oblita
High-Throughput Nucleotide Sequencing
Holotrichia
Insect Proteins - chemistry
Insect Proteins - genetics
Insect Proteins - metabolism
Insects
Invertebrate Microbiology
Larva - chemistry
Larva - genetics
Larva - metabolism
Larva - microbiology
Larvae
Lepidoptera
Liquid chromatography
Mass spectrometry
Mass spectroscopy
Membrane vesicles
Midgut
Protein Binding
Proteins
Receptors
Ribonucleic acid
RNA
Serine
Serine proteinase
Tandem Mass Spectrometry
Toxins
Transcriptome
Transferrin
Transferrins
Tribolium castaneum
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Summary:The strain HBF-18 (CGMCC 2070), containing two genes ( -like and ), is toxic to larvae. Both Cry8-like and Cry8Ga proteins are active against this insect pest, and Cry8-like is more toxic. To analyze the characteristics of the binding of Cry8-like and Cry8Ga proteins to brush border membrane vesicles (BBMVs) in larvae, binding assays were conducted with a fluorescent DyLight488-labeled Cry8-like toxin. The results of saturation binding assays demonstrated that Cry8-like bound specifically to binding sites on BBMVs from , and heterologous competition assays revealed that Cry8Ga shared binding sites with Cry8-like. Furthermore, Cry8-like-binding proteins in the midgut from larvae were identified by pulldown assays and liquid chromatography-tandem mass spectrometry (LC-MS/MS). In addition, the midgut transcriptome was assembled by high-throughput RNA sequencing and used for identification of Cry8-like-binding proteins. Eight Cry8-like-binding proteins were obtained from pulldown assays conducted with BBMVs. The LC-MS/MS data for these proteins were successfully matched with the transcriptome, and BLASTX results identified five proteins as serine protease, transferrin-like, uncharacterized protein LOC658236 of , ATPase catalytic subunit, and actin. These identified Cry8-like-binding proteins were different from those confirmed previously as receptors for Cry1A proteins in lepidopteran insect species, such as aminopeptidase, alkaline phosphatase, and cadherin. is one of the main soil-dwelling pests in China. The larvae damage the roots of crops, resulting in significant yield reductions and economic losses. is difficult to control, principally due to its soil-dwelling habits. In recent years, some Cry8 toxins from were shown to be active against this pest. Study of the mechanism of action of these Cry8 toxins is needed for their effective use in the control of and for their future utilization in transgenic plants. Our work provides important basic data and promotes understanding of the insecticidal mechanism of Cry8 proteins against larvae.
ISSN:0099-2240
1098-5336
DOI:10.1128/AEM.00541-17