A key gene of the RNA interference pathway in the black tiger shrimp, Penaeus monodon: identification and functional characterisation of Dicer-1.
RNA interference (RNAi) is an evolutionarily conserved mechanism by which double-stranded RNA (dsRNA) initiates post-transcriptional silencing of homologous genes. Here we report the amplification and characterisation of a full length cDNA from black tiger shrimp (Penaeus monodon) that encodes the bidentate RNAase III Dicer, a key component of the RNAi pathway. The full length of the shrimp Dicer (Pm Dcr1) cDNA is 7629bp in length, including a 5' untranslated region (UTR) of 130bp, a 3' UTR of 77bp, and an open reading frame of 7422bp encoding a polypeptide of 2473 amino acids with an estimated molecular mass of 277.895kDa and a predicted isoelectric point of 4.86. Analysis of the deduced amino acid sequence indicated that the mature peptide contains all the seven recognised functional domains and is most similar to the mosquito (Aedes aegypti) Dicer-1 sequence with a similarity of 34.6%. Quantitative RT-PCR analysis showed that Pm Dcr1 mRNA is most highly expressed in haemolymph and lymphoid organ tissues (P<0.05). However, there was no correlation between Pm Dcr1 mRNA levels in lymphoid organ and the viral genetic loads in shrimp naturally infected with gill-associated virus (GAV) and Mourilyan virus (P>0.05). Treatment with synthetic dsRNA corresponding to Pm Dcr1 sequence resulted in knock-down of Pm Dcr1 mRNA expression in both uninfected shrimp and shrimp infected experimentally with GAV. Knock-down of Pm Dcr1 expression resulted in more rapid mortalities and higher viral loads. These data demonstrated that Dicer is involved in antiviral defence in shrimp.
Su
Jianguo
J
CSIRO Livestock Industries, Queensland Bioscience Precinct, 306 Carmody Road, St Lucia, Queensland, Australia.
Oanh
Dang T H
DT
Lyons
Russell E
RE
Leeton
Lisa
L
van Hulten
Marielle C W
MC
Tan
Siok-Hwee
SH
Song
Linsheng
L
Rajendran
K V
KV
Walker
Peter J
PJ
D000595:Amino Acid Sequence; D000818:Animals; D018076:DNA, Complementary / Q000737:chemistry; D015870:Gene Expression / Q000187:drug effects / Q000276:immunology*; D020869:Gene Expression Profiling / Q000662:veterinary; D023061:Gene Order; D008969:Molecular Sequence Data; D033561:Penaeidae / Q000187:drug effects / Q000276:immunology* / Q000821:virology*; D010802:Phylogeny; D020365:RNA Helicases / Q000032:analysis / Q000096:biosynthesis / Q000235:genetics*; D012330:RNA, Double-Stranded / Q000494:pharmacology; D012333:RNA, Messenger / Q000032:analysis / Q000096:biosynthesis; D046828:Roniviridae / Q000276:immunology* / Q000472:pathogenicity*; D016415:Sequence Alignment / Q000662:veterinary; D017386:Sequence Homology, Amino Acid; D014018:Tissue Distribution; D019562:Viral Load / Q000662:veterinary
D018076:DNA, Complementary; D012330:RNA, Double-Stranded; D012333:RNA, Messenger; D020365:RNA Helicases