Scientia Agricultura Sinica ›› 2014, Vol. 47 ›› Issue (10): 1947-1955.doi: 10.3864/j.issn.0578-1752.2014.10.008

• PLANT PROTECTION • Previous Articles     Next Articles

Cloning and Expression Analysis of a Carboxylesterase Gene BdCAREB1 from Bactrocera dorsalis

 SHEN  Guang-Mao, DOU  Wei, WANG  Jin-Jun   

  1. Key Laboratory of Entomology and Pest Control Engineering, College of Plant Protection, Southwest University, Chongqing 400716
  • Received:2013-10-09 Online:2014-05-20 Published:2013-11-20

Abstract: 【Objective】Based on the cloning of a carboxylesterase gene of Bactrocera dorsalis, the expressions of this gene among different developmental stages, tissues, and under insecticide stimulation were analyzed. Combined with previous data of enzyme activity assay and synergist analysis, the objectives of this study are to clarify the reaction of carboxylesterase when stimulated by β-cypermethrin, and lay a foundation for the future study of the mechanism that how carboxylesterase works in the detoxification of pyrethroid insecticides.【Method】From the transcriptome data of B. dorsalis, a partial sequence of carboxylesterase gene was screened out by using homologous blast, and the full sequence was cloned by using RACE technology. The ORF, deduced amino acid sequence, and molecular weight were predicted, and a phylogenetic tree with carboxylesterase genes from other insects was constructed by using maximum likelihood method to clarify its molecular characterization. RNA was extracted from different developmental stages and the midgut, Malpighian tubules, fat body, trachea of the 3rd instar larvae. Based on the reference evaluation, qPCR was used for the expression analysis of different developmental stages, tissues, and stimulated by insecticide. 【Result】A partial sequence of 1 028 bp was found in the transcriptome data. The results of 5′ and 3′ amplification were 1 073 and 625 bp, respectively. After sequence assemble and verification, the full-length of this gene was 1 872 bp, contained a complete ORF of 1 710 bp, and encoding a protein of 569 amino acids with a predicted molecular mass of 63.3 kD. It was named as BdCAREB1 and submitted to GenBank with an accession number of KF539980. The phylogenetic tree of BdCAREB1 showed high homology with carboxylesterase of Diptera. The qPCR result of different developmental stages of the 3rd instar larvae showed BdCAREB1 was highly expressed in the 3rd instar larvae, followed by the adults, and it was the lowest in the eggs. Among different tissues, the BdCAREB1 was highly expressed in fat body. The expressions between midgut and Malpighian tubules were quite the same, and in trachea, the expression was the lowest. After the insects were fed on a diet containing 0.33 μg?g-1 (insecticide/diet) β-cypermethrin, the expression of BdCAREB1 was significantly up-regulated in larvae and fat body. 【Conclusion】The specific expressions of BdCAREB1 were identified in different developmental stages and tissues, which were highly enriched in the larvae and fat body. It showed inducibility by the stimulation of β-cypermethrin. Combined with previous data of enzyme activity assay and synergist analysis, BdCAREB1 may be involved in the detoxification of β-cypermethrin.

Key words: Bactrocera dorsalis , carboxylesterase , gene expression , fat body , detoxification

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