Scientia Agricultura Sinica ›› 2015, Vol. 48 ›› Issue (12): 2364-2373.doi: 10.3864/j.issn.0578-1752.2015.12.009

• PLANT PROTECTION • Previous Articles     Next Articles

Identification and Functional Characterization of TRPA1 in Apolygus lucorum (Meyer-Dür)

FU Ting, YANG Ting, LIU Yang, WANG Gui-rong   

  1. State Key Laboratory for Biology of Plant Diseases and Insect Pests, Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing 100193
  • Received:2015-01-19 Online:2015-06-16 Published:2015-06-16

Abstract: 【Objective】 The objective of this study is to obtain the TRPA1 gene in Apolygus lucorum,characterize its expression profiles in different tissues of adult, illustrate its functions through heterologous expression system of Xenopus oocytes, and further to explain the molecular mechanism of thermosensation in A. lucorum.【Method】The transcriptome of A. lucorum was sequenced and analyzed, and several cDNA fragments encoding A. lucorum TRPA1 were identified. And then specific primers for RT-PCR and RACE were designed. The full-length cDNA sequence encoding AlucTRPA1 was cloned via RACE and RT-PCR techniques. The cDNA sequence encoding TRPA1 gene was analyzed by DNAMAN software and BLAST. Then ankyrin repeats and transmembrane domains were predicted with SMART and TMpred. The phylogenetic tree of TRPs in insects was constructed to analyze the evolutionary relationship between A. lucorum and other insects. RT-PCR was performed to detect the relative-expression of TRPA1 in different tissues of the adults. Two-electrode voltage clamp electrophysiological recording was introduced to demonstrate the function of TRPA1 in vitro. 【Result】 Four fragment sequences of the TRPA1gene were found according to the results of transcriptome analysis. The full-length sequence of TRPA1 gene named AlucTRPA1 was further obtained using RACE and RT-PCR techniques, which is 3 672 bp and encodes 1 223 amino acids. AlucTRPA1 contained 6 conserved transmembrane domains and 16 ankyrin repeats, which predicted by SMART and TMpred. The results of multiple alignment and phylogenetic tree analysis showed that TRPA of insects contains TRPA1, Painless, Pyrexia and Water witch, and AlucTRPA1 clustered into the branch of TRPA1. AlucTRPA1 is highly similar to the homologs from other insects, especially the pea aphids (Acyrthosiphon pisum), which also belongs to the Hemiptera, with the identity of 65.1%. Expression profiles showed that AlucTRPA1 was expressed in antennae at the highest level among different tissues. In the Xenopus-based functional study, AlucTRPA1 could be activated directly by an increasing temperature from 20 to 40 and didn’t show adaption after repeated temperature stimuli. 【Conclusion】 AlucTRPA1ors. expressed in all tested tissues, but highest in antennae. AlucTRPA1 could be activated directly by an increasing temperature from 20 to 40, indicating that AlucTRPA1 is a direct thermal sensor in A. lucorum and mediates their temperature sensing behavi

Key words: Apolygus lucorum, TRPA1, temperature, Xenopus oocytes, the two-electrode voltage-clamp

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