title: Assembling of Holotrichia parallela (dark black chafer) midgut tissue transcriptome and identification of midgut proteins that bind to Cry8Ea toxin from Bacillus thuringiensis.
abstract: Holotrichia parallela is one of the most severe crop pests in China, affecting peanut, soybean, and sweet potato crops. Previous work showed that Cry8Ea toxin is highly effective against this insect. In order to identify Cry8Ea-binding proteins in the midgut cells of H. parallela larvae, we assembled a midgut tissue transcriptome by high-throughput sequencing and used this assembled transcriptome to identify Cry8Ea-binding proteins by liquid chromatography-tandem mass spectrometry (LC-MS/MS). First, we obtained de novo sequences of cDNAs from midgut tissue of H. parallela larvae and used available cDNA data in the GenBank. In a parallel assay, we obtained 11 Cry8Ea-binding proteins by pull-down assays performed with midgut brush border membrane vesicles. Peptide sequences from these proteins were matched to the H. parallela newly assembled midgut transcriptome, and 10 proteins were identified. Some of the proteins were shown to be intracellular proteins forming part of the cell cytoskeleton and/or vesicle transport such as actin, myosin, clathrin, dynein, and tubulin among others. In addition, an apolipophorin, which is a protein involved in lipid metabolism, and a novel membrane-bound alanyl aminopeptidase were identified. Our results suggest that Cry8Ea-binding proteins could be different from those characterized for Cry1A toxins in lepidopteran insects.
authors.affiliation: State Key Laboratory of Biology for Plant Diseases and Insect Pests, Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing, 100193, People's Republic of China.
authors.forename: Changlong
authors.initials: C
authors.lastname: Shu
authors.forename: Shuqian
authors.initials: S
authors.lastname: Tan
authors.forename: Jiao
authors.initials: J
authors.lastname: Yin
authors.forename: Mario
authors.initials: M
authors.lastname: Soberón
authors.forename: Alejandra
authors.initials: A
authors.lastname: Bravo
authors.forename: Chunqing
authors.initials: C
authors.lastname: Liu
authors.forename: Lili
authors.initials: L
authors.lastname: Geng
authors.forename: Fuping
authors.initials: F
authors.lastname: Song
authors.forename: Kebin
authors.initials: K
authors.lastname: Li
authors.forename: Jie
authors.initials: J
authors.lastname: Zhang
mesh_terms: D000818:Animals; D010367:Arachis / Q000469:parasitology; D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins / Q000378:metabolism*; D002498:Centrifugation; D002681:China; D002853:Chromatography, Liquid; D001517:Coleoptera / Q000187:drug effects*; D004731:Endotoxins / Q000378:metabolism*; D041981:Gastrointestinal Tract / Q000502:physiology; D020869:Gene Expression Profiling; D006460:Hemolysin Proteins / Q000378:metabolism*; D059014:High-Throughput Nucleotide Sequencing; D027723:Ipomoea batatas / Q000469:parasitology; D007814:Larva / Q000187:drug effects; D011485:Protein Binding; D013025:Glycine max / Q000469:parasitology; D053719:Tandem Mass Spectrometry
chemical_list: D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins; D004731:Endotoxins; D006460:Hemolysin Proteins; C030956:insecticidal crystal protein, Bacillus Thuringiensis