Molecular characteristics, mRNA expression, and alternative splicing of a ryanodine receptor gene in the oriental fruit fly, Bactrocera dorsalis (Hendel)

PLoS One. 2014 Apr 16;9(4):e95199. doi: 10.1371/journal.pone.0095199. eCollection 2014.

Abstract

Ryanodine receptors (RyRs) are a distinct class of ligand-gated channels controlling the release of calcium from intracellular stores. The emergence of diamide insecticides, which selectively target insect RyRs, has promoted the study of insect RyRs. In the present study, the full-length RyR cDNA (BdRyR) was cloned and characterized from the oriental fruit fly, Bactrocera dorsalis (Hendel), a serious pest of fruits and vegetables throughout East Asia and the Pacific Rim. The cDNA of BdRyR contains a 15,420-bp open reading frame encoding 5,140 amino acids with a predicted molecular weight of 582.4 kDa and an isoelectric point of 5.38. BdRyR shows a high level of amino acid sequence identity (78 to 97%) to other insect RyR isoforms. All common structural features of the RyRs are present in the BdRyR, including a well-conserved C-terminal domain containing consensus calcium-binding EF-hands and six transmembrane domains, and a large N-terminal domain. Quantitative real-time PCR analyses revealed that BdRyR was expressed at the lowest and highest levels in egg and adult, respectively, and that the BdRyR expression levels in the third instar larva, pupa and adult were 166.99-, 157.56- and 808.56-fold higher, respectively, than that in the egg. Among different adult body parts, the highest expression level was observed in the thorax compared with the head and abdomen. In addition, four alternative splice sites were identified in the BdRyR gene, with the first, ASI, being located in the central part of the predicted second spore lysis A/RyR domain. Diagnostic PCR analyses revealed that alternative splice variants were generated not only in a tissue-specific manner but also in a developmentally regulated manner. These results lay the foundation for further understanding the structural and functional properties of BdRyR, and the molecular mechanisms for target site resistance in B. dorsalis.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Alternative Splicing*
  • Amino Acid Sequence
  • Animals
  • Conserved Sequence
  • DNA, Complementary / genetics
  • DNA, Complementary / metabolism
  • Gene Expression Regulation
  • Insect Proteins / genetics*
  • Insect Proteins / metabolism
  • Isoelectric Point
  • Molecular Sequence Data
  • Molecular Weight
  • Open Reading Frames
  • Phylogeny
  • Protein Structure, Tertiary
  • RNA, Messenger / genetics*
  • RNA, Messenger / metabolism
  • Ryanodine Receptor Calcium Release Channel / genetics*
  • Ryanodine Receptor Calcium Release Channel / metabolism
  • Sequence Alignment
  • Sequence Homology, Amino Acid
  • Tephritidae / classification
  • Tephritidae / genetics*
  • Tephritidae / metabolism

Substances

  • DNA, Complementary
  • Insect Proteins
  • RNA, Messenger
  • Ryanodine Receptor Calcium Release Channel

Grant support

This research was supported in part by the Fundamental Research Funds for Central Universities, China (XDJK2012C076), the Doctor Foundation Project of Southwest University (SWU112002), China Postdoctoral Science Foundation (2013M540693) and Fundamental and Advanced Research Program of Chongqing (CSTC, 2013jcyjA80020). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.