Cloning and RNAi-mediated functional characterization of MaLac2 of the pine sawyer, Monochamus alternatus

Insect Mol Biol. 2008 Jun;17(3):303-12. doi: 10.1111/j.1365-2583.2008.00803.x.

Abstract

Laccase, a member of a group of proteins collectively known as multicopper oxidases, is hypothesized to play an important role in insect cuticle sclerotization by oxidizing catechols in the cuticle to their corresponding quinones, which then catalyze protein cross-linking reactions. Laccase 2 has been proved as the gene required for beetle cuticle tanning through RNA interference (RNAi) experiments on red flour beetle Tribolium castaneum. The pine sawyer beetle, Monochamus alternatus (Coleoptero: Cerambycidae) is the insect serving as a major vector of the pinewood nematode, Bursaphelenchus xylophilus, which is the causative agent for pine wilt disease. The cDNA of MaLac2 was cloned from the insect in this study. The conceptual amino-acid sequence deduced was much conserved with other known insect laccases, particularly with the enzyme of Tribolium castaneum. Injection in hemolymph of pine sawyer larva of dsRNA targeting the laccase 2 mRNA leads to important alterations of the tanning, hardening and sclerotization of the pupal and adult cuticles. Defaults appear in a dose-dependent manner and high loads of dsRNA are lethal. The decrease of the endogenous laccase 2 mRNA affects the procuticle which is thinner and without the characteristic piling up of successive layers. The observations reinforce the role of laccase 2 as an essential phenoloxidase for making cuticle.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Animals
  • Base Sequence
  • Cloning, Molecular
  • Coleoptera / genetics*
  • Coleoptera / growth & development
  • Coleoptera / ultrastructure
  • DNA, Complementary / genetics
  • Gene Expression Regulation, Developmental
  • Insect Proteins / chemistry
  • Insect Proteins / genetics*
  • Insect Proteins / metabolism
  • Integumentary System / anatomy & histology
  • Molecular Sequence Data
  • Phenotype
  • Phylogeny
  • Pigmentation
  • Pinus / parasitology*
  • RNA Interference*
  • Sequence Analysis, DNA

Substances

  • DNA, Complementary
  • Insect Proteins

Associated data

  • GENBANK/EU093075