关键词: 小菜蛾, Bt毒素, Polycalin, 基因克隆, 基因表达, Cry1Ac毒素

Abstract: 【Aim】 As a newly found receptor of Bt toxin, polycalin is considered to be related to the resistance of insects to Bt toxin. This study aims to clarify the relationship between polycalin and the Bt Cry1Ac toxin in Plutella xylostella. 【Methods】 The full-length cDNA sequence of polycalin gene was cloned from the midgut of the 3rd instar larvae of P. xylostella by PCR and RACE techniques. The expression levels of the polycalin gene in different developmental stages and different tissues of the 4th instar larvae were determind by quantitative real-time PCR. The expression levels of the polycalin gene in the 3rd instar larvae fed with different concentrations of Cry1Ac toxin were also compared. The brush border membrane vesicles (BBMV) proteins in larval midgut were separated, and monoclonal antibodies were prepared by peptide synthesis technique. The polycalin and its binding characteristics to Cry1Ac toxin were identified by using Western blot and Ligand blot. 【Results】 The full-length cDNA (GenBank accession no.: MF138149) of polycalin gene obtained from P. xylostella is 9 102 bp in length, with the open reading frame of 8 778 bp, encoding 2 925 amino acids. The predicted molecular weight and isoelectric point of the encoded protein are 326.38 kD and 4.39, respectively. The putative protein sequence contains an N-terminal signal peptide of 20 amino acid residues, 14 N-linked and 67 O-linked glycosylation sites, with six lipocalin signatures, six lipocalin hits and 13 lipocalin like structures. There are a glycoprotein cleavage-activation site between the 20th and 21th (TSG-QVV) amino acid residues, and a GPI anchor signal peptide with two amino acid residues at the C-terminus. The polycalin has the feature of Bt toxin receptor. Developmental expression profiles revealed that the polycalin gene had higher expression level in larva than in pupa and adult, with the highest expression level in the 3rd instar larva. Tissue expression profiles revealed that the polycalin gene was expressed in the head, thorax and abdomen of the 4th instar larvae, with the highest expression level in the abdomen. The expression of the polycalin gene in the 3rd instar larvae fed with different concentrations of Cry1Ac toxin was dramatically decreased as compared with that of the control (fed with PBS). The higher the concentration of Cry1Ac toxin, the lower the expression level of the polycalin gene. The polycalin in the brush border membrane vesicles (BBMV) of the midgut of P. xylostella was approximately 300 kD by Western blot analysis. The Ligand blot result confirmed that this polycalin protein could bind to Cry1Ac toxin. 【Conclusion】 This study verified that the polycalin from P. xylostella could bind to Bt toxin, and provides useful information for further study on the action mechanisms of Bt against insects and pest control by utilization of Bt.

Key words: Plutella xylostella, Bt toxin, polycalin, gene cloning, gene expression, Cry1Ac toxin