Table 1.
Primer sequences for amplification.
Fig 1.
The gene information of PmChk1.
(A) Full-length cDNA of PmChk1 (schematic). The deduced amino-acid sequence is depicted underneath the nucleotide sequence. Initiation code (ATG) and termination code (TAA) are denoted by boxes. Polyadenylation signal sequence (AATAAA) is emboldened. (B) Multiple alignment of the deduced amino-acid sequences of Chk1 from P. monodon and other species. A sequence logo denoting similarity is shown at the top of alignments, and numbers of amino acids are shown on the right-hand side of alignments. GenBank numbers of Chk1 were: P. monodon: KU958380; Homo sapiens: AAC51736.1; Poeciliopsis prolifica: JAO88875.1; Mus musculus: AAC53334.1; Daphnia pulex: AGN95867.1; Melipona quadrifasciata: KOX69887.1; Cyphomyr mexcostatus: KYN05919.1. The S-TKc domain is indicated with a red box. (C) Neighbor-joining phylogenetic tree of E2F-2s based on amino-acid sequences. Confidence in each node was evaluated by 2000 bootstrap replicates using Mega v5.03. (D) Comparison of the genomic DNA sequence encoding Chk1 in P. monodon. Green-shaded rectangles denote exons, gray horizontal lines denote introns, and the numbers reflect the length (in bp) of exons and introns.
Fig 2.
Nucleotide sequence of PmChk1 demonstrating the 5ʹ upstream genomic sequence.
Numbering of the nucleotide sequence commences from the transcription start site (+1) (arrow) and proceeds as positive numbers in the 3ʹ direction and as negative numbers in the 5ʹ direction. The putative binding sequence motifs for transcription factors and ribosomal promoter site are underlined.
Fig 3.
Relative expression of PmChk1 in different tissues at stage II (chromatin nucleolus).
Relative expression of PmChk1 in different tissues according to quantitative real-time PCR using EF-1α as an internal reference. Vertical bars represent the mean ± SD (n = 3 for each group). Different letters above the vertical bars denote significant differences (P < 0.05).
Fig 4.
Relative expression of PmChk1 in various conditions.
(A) Relative expression of PmChk1 in various developmental stages. The developmental stages were spawned egg, nauplius, protozoea I–III, mysis I–III, and post-larval. Relative expression of PmChk1 in developmental stages was measured by quantitative real-time PCR with EF-1α employed as an internal reference. (B) Relative expression of PmChk1 in ovaries at different developmental stages. Relative expression of PmChk1 in ovaries at different developmental stages [I (ovogonium); II (chromatin nucleolus); III (perinucleolus); IV (yolky); V (cortical rod)] measured by qRT-PCR using EF-1α as an internal reference. (C) Expression of PmChk1 mRNA after stimulation with 5-HT or DA. Relative expression of PmChk1 mRNA in ovaries after treatment with 5-HT or DA. (D) Expression of PmChk1 mRNA after eyestalk ablation. Relative expression of PmChk1 mRNA in ovaries after eyestalk ablation. Vertical bars represent the mean ± SD (n = 3 for each group). Different letters above the vertical bars denote significant differences (P < 0.05).
Fig 5.
Relative expression of PmChk1 in the ovaries and hepatopancreas of P. monodon after dsRNA-RBL and dsRNA-p53 treatment.
(A) Relative expression of PmChk1 in ovaries. (B) Relative expression of PmChk1 in the hepatopancreas. (C) Relative expression of PmChk1 in ovaries. (D) Relative expression of PmChk1 in the hepatopancreas. Ovary and hepatopancreas tissues harvested from P. monodon injected with dsRNA-RBL were compared with respect to expression of PmChk1 mRNA (relative to EF-1α) using the Student’s t-test. Vertical bars represent the mean ± SD (n = 3 for each group). Significant differences from controls are denoted: **P < 0.01, *P < 0.05.
Fig 6.
Expression of PmChk1 protein and gene after dsRNA-Chk1 injection.
(A) Expression of PmChk1 protein in ovaries after dsRNA-Chk1 injection. Lane M = pre-stained molecular-weight marker. (B) Relative expression of PmChk1 in the ovaries of P. monodon after dsRNA-Chk1 treatment. (C) Relative expression of PmChk1 in the hepatopancreas of P. monodon after dsRNA-Chk1 treatment. Vertical bars represent the mean ± SD (n = 3 for each group). Significant differences from controls are denoted: **P < 0.01, *P < 0.05.
Fig 7.
Relative expression of PmCDC2 and PmCyclin B in the ovaries and hepatopancreas of P. monodon after dsRNA-Chk1 treatment.
(A) Relative expression of PmCDC2 in ovaries. (B) Relative expression of PmCDC2 in the hepatopancreas. (C) Relative expression of PmCyclin B in ovaries. (D) Relative expression level of PmCyclin B in the hepatopancreas. Vertical bars represent the mean ± SD (n = 3 for each group). Significant differences from controls are denoted: **P < 0.01, *P < 0.05.
Fig 8.
In situ detection of PmChk1 hybridization.
The hepatopancreas was harvested after dsRNA-Chk1 administration. Brown dots denote positive reactions (arrows). A represents a negative control. B represents the collected hepatopancreas after dsRNA-GFP injection. C represents the collected hepatopancreas after dsRNA-Chk1 injection. Scale bar = 30.
Fig 9.
Chk1 activity and the GSI of P. monodon after dsRNA-Chk1 injection.
(A) Chk1 activity after injection of dsRNA-Chk1 and dsRNA-GFP. Vertical bars represent the mean ± SD (n = 3 for each group). Significant differences from controls are denoted: *P < 0.05. (B) The GSI (ovary weight/body weight × 100) of P. monodon after injection of dsRNA-GFP, dsRNA-Chk1, or PBS.