(Promega, Madison, WI, USA), 1 ml dNTP (10 mM each; Promega), 1 ml recombinant RNasin ribonuclease inhibitor (Promega), 5 ml M- MLV 5 6 reaction buffer (Promega) and 5 ml DEPC-treated water were mixed and incubated at 42uC for 60 min. The reaction was terminated by heating at 70uC for 15 min. The resulting cDNAs were stored at _20uC until further use. The PjCaspase cDNA was amplified using two PjCaspase gene- specific primers (59-ATGGACGAGGTAATCCAG-39 and 59- TCACTGTGGGGCGGAG-39) [16]. As a control, the shrimp b- actin gene was included in the amplification with primers 59-AT- GTGT GACGACGAAGTAGC-39 and 59-GAAGCACTTCC- TGTGAACGA-39. PCR was conducted with an initial denatur- ation step of 5 min at 94uC, followed by 30 cycles of 30 s at 94uC, 30 s at 50uC and 60 s at 72uC. All the PCRs were performed with Pfx DNA polymerase (Invitrogen, Carlsbad, CA, USA). PCR products were purified (Omega Bio-tek, Norcross, GA, USA) and cloned into pMD18-T vector. Twenty of the PjCaspase cDNA clones on a plate were selected randomly for sequencing. Statistical analysis of sequences To infer the diversification of variations in the PjCaspase cDNA sequences, as well as in the actin cDNA sequences, the criterion reported by Zhang et al. [21] was used to draw out the reliable sequences as the sources which were considered as original and un-divergent. The total variation number in each cluster was minimized. Southern blot analysis Genomic DNA was prepared from shrimp hemolymph using a SQ tissue DNA Kit (Omega Bio-tek, Norcross, GA, USA) according to the manufacturer’s instructions. Briefly, the fresh shrimp hemolymph was homogenized thoroughly in the WTL buffer using a microfuge tube pestle. After adding proteinase K solution (20 mg/ml), the homogenized mixture was incubated at 60uC for 3 hours. Thereafter, RNase A solution was added to the mixture and incubated for 15 min at 37uC. The PCP buffer was added and vortexed vigorously for 30 seconds, followed by incubation on ice for 5 min and centrifugation at 13,000 g for 3 min at room temperature. Subsequently the supernatant was mixed with 100% isopropanol and centrifuged at 14,000 g for 1 min at room temperature. After washing with 70% ethanol several times, the genomic DNA was resuspended in sterile water and stored at 2u–8uC for later use. The genomic DNA extracted from shrimp hemolymph was digested with DraI or EcoRI, respectively, and then separated by electrophoresis on a 0.7% agarose gel in 0.56TBE buffer (90 mM Tris-boric acid, 2 mM EDTA, PH 8.0). The separated DNA was transferred to a nylon membrane (Amersham Biosciences, Fair- field, CT, USA). The blots were probed with a DIG-labeled PjCaspase gene that could detect all mutations of PjCaspase. For the labeling of PjCaspase gene, the full-length gene was amplified by PCR and randomly labeled with DIG. The DIG labeling and detection were performed following the protocol of the DIG High Prime DNA Labeling and Detection Starter Kit (Roche, Grenzacherstrasse, Basel, Switzerland). Synthesis of siRNA The siRNAs used in this study consisted of 21-nucleotide double-stranded RNAs, each strand of which contained a 17,22- nucleotide target sequence and a two-uracil (U) overhang at the 39 end [28]. Based on the different sequences between the caspases from WSSV- resistant shrimp and virus-free shrimp, 6 fragments of the PjCaspase gene being prognosticated to be involved in shrimp Gene Sequence Diversity for Antiviral Immunity antiviral defense were selected for RNAi assays. The siRNAs were designed according to the sequences of 6 fragments of the PjCaspase gene. The sequences of siRNAs respectively targeting the 6 fragments of PjCaspase gene were as follows: fragment 159-TTGGATCCTACCGTAGGAGTG-39 fragment 259-T TGAGGTACTCCTGCAGGCT T-39 fragment 359-CCTCTTCACAGGCCGAGG ATT-39 fragment 459-ATAGAACT GGTATTCAGGCTT-39 fragment 559-TTAGCACCTTCAAGTCTGAATT-39 fragment 659-CGAAAGGTGGTCAGG AATGGTT-39 The siRNA sequence of fragment 3 (siRNA3), showing the highest apoptotic activity, was randomly mutated at one nucleotide, resulting in siRNA 3-mutation (59-CCTATTCACA- GGCCGAGGATT-39). The sequence of siRNA having RNAi capacity to all PjCaspase mRNA molecules was 59-GTTATGAA- GTGCTACAG-39 (PjCaspase siRNA). The siRNAs were synthesized in vitro, using a commercially available kit, according to the manufacturer’s recommendations (TaKaRa, Japan). The formation of double-stranded RNAs was monitored by determining the size shift in agarose gel electropho- resis. The synthesized siRNAs were dissolved in siRNA buffer (50 mM Tris-HCl, 100 mM NaCl, PH 7.5) and quantified spectrophotometrically. RNAi assay of shrimp in vivo The RNAi assays were conducted with the following 9 treatments: physiological saline only; WSSV (105 virion copies/ ml) only; WSSV (105 virion copies/ml)+siRNA 3 (24 mM); WSSV (105 virion copies/ml)+siRNA 3-mutation (24 mM); WSSV (105 virion copies/ml)+ PjCaspase siRNA (24 mM); Vibrio parahaemoly- ticus (106 copies/ml) only; Vibrio parahaemolyticus (106 copies/ ml)+siRNA 3; Vibrio parahaemolyticus (106 copies/ml)+siRNA 3- mutation and Vibrio parahaemolyticus (106 copies/ml)+ PjCaspase siRNA). The WSSV, Vibrio parahaemolyticus and/or siRNA dissolved in physiological saline solution were delivered into the shrimp by simultaneous injection into the juncture area between the third and forth abdominal section at 0.1 ml/shrimp, using a syringe with a 29-gauge needle. At different time intervals p.i., the shrimp hemolymph was collected from the ventral sinus by inserting a 26- gauge needle containing heparin sodium (500 U/ml). Three shrimp specimens from each treatment, selected at random, were subjected to Northern blot and apoptosis assays, including detection of caspase-3 activity and DAPI staining of shrimp hemocytes. All assays were conducted in biological triplicate. Synthesis of PjCaspase mRNA containing fragment 3 and its over-expression in shrimp The synthesis of PjCaspase mRNA containing fragment 3 and its over-expression in shrimp were conducted as described previously (Wu et al., 2008). To synthesize the PjCaspase mRNA containing fragment 3, the PjCaspase gene was divided into two segments, segment 1 (from the 59 terminal of the PjCaspase gene to fragment 3) and segment 2 (from fragment 3 to the 39 terminal of the PjCaspase gene). Segment 1 was amplified by PCR with primers 59- GATCA CTAATACGACTCACTATAGGGATGGACGAGG- TAATCCAGGT-39 (T7 promoter, underlined) and 59-CCTC- TTCACAGGCCGAGGATT-39 (sequence of fragment 3). Seg- ment 2 was amplified by PCR with primers 59-AATCCTCGG- CCTGTGAAGAGG-39 (complementary sequence of fragment 3) and 59-TCACTGTGGGGCGGAGTGGA-39. After amplifica- tions, segments 1 and 2 were denatured at 99uC and annealed. The annealed DNAs were amplified by PCR with primers 59- GATCACTAATACGACTCACTATAGG-39 and 59-TCACTG- PLoS ONE | www.plosone.org 8 September 2011 | Volume 6 | Issue 9 | e24955