developmental stages including nauplius , postlarva and juvenile were collected for RNA extraction. Total RNA was isolated by TRIZOL Reagent (Invitrogen, Carlsbad, CA), following the method described of previous work [22]. Total RNA concentra- tions were determined by a spectrophotometer [23]. Protein extraction Cytoplasmic and nuclear protein were extracted as previously described [11]. Briefly, 150 mg tissue was homogenized in buffer A (10 mM HEPES, pH 7.9, 10 mM KCl, 0.1 mM EDTA) contained protease inhibitor (1 mM DTT, 0.5 mM PMSF, 10 mg/ml leupeptin) and 40 ml of 10% NP40, incubated at room temperature. Cell debases and un-break cell was separated by centrifugation follow by another centrifugation for nuclear and cytosolic protein separation. Nuclear pellet was resuspened by buffer B (20 mM HEPES, pH 7.9, 0.4 M NaCl, 1 mM EDTA, 10% glycerol) as the nuclear protein fraction. Protein concentra- tion was determined by using BCA method (Pierce). Expression of LvDBP23 in different tissues Total RNA samples were treated with DNaseI to degrade any possible genomic DNA. Briefly, total RNA (1.5 mg) was added in a 10 ml reaction containing 1 ml 106 buffer (200 mM Tris-HCl, pH 8.4, 20 mM MgCl2, 500 mM KCl), 1.5 ml DNase I (1.5 U, Invitrogen, Carlsbad, CA) at 37uC for 15 min, followed by inactivation with 25 mM EDTA at 65uC for 10 min. Reverse transcription (RT) was performed using 1.5 mg RNA, 0.5 mg Oligo (dT) 18 mers, 1.0 mM dNTP, 4 ml of 56 reaction buffer and 40 U of M-MuLV reverse transcriptase (Fermentas, Burlington, Cana- da) at 37uC for 60 min. The reaction was stopped by heating at 70uC for 10 min. For PCR amplification, two oligonucleotide primers, B47_F2: 59 GCGACTCTGGCTTGGGCGTTGAC 39 and B47_R2: 59CTGACGTGGGCGACATCGTTG GG 39 were designed based on the nucleotide sequence corresponding to the nucleotide positions the position 168–190 (B47_F2) and 609–635 (B47_R2) of L. vannamei LvDBP23 cDNA, respectively. A set of control, one pair of elongation factor 1a (EF-1a) primer, EF-1_F and EF-1_R [24] was used during the RT-PCR amplification. PCR reactions were conducted for 40 cycles with denature at 94uC for 30 sec, annealing at 60uC for 30 sec, and extension at 72uC for 30 sec in a reaction buffer consisting of 10 mM Tris (pH 8.3), 50 mM KCl, 200 M dNTPs, 0.2 mM of each primer, and 2.5 U of Taq Polymerase (Biolabs Inc., Ipswich, MA) in a total volume of 50 ml. To visualize the amplified products, PCR reaction was electrophoresed through 1.5% agarose gel containing 0.025% ethidium bromide and observed under the UV light. References 1. Wyban J (2009) World Shrimp Farming Revolution: Industry Impact of Domestication, Breeding and Widespread Use of Specific pathogen-free Penaeus vannamei. Proceedings of the Special Session on Sustainable Shrimp Farming, World Aquaculture 2009: 12–21. 2. Baylies MK, Bate M, Gomez MR (1998) Myogenesis: a view from Drosophila. Cell 93: 921–927. 3. Perry RL, Rudnick MA (2000) Molecular mechanisms regulating myogenic determination and differentiation. Front Biosci 5: D-750–D767. 4. Cesar JRD, Zhao B, Malecha S, Ako H, Yang J (2006) Morphological and biochemical changes in the muscle of the marine shrimp Litopenaeus vannamei during the molt cycle. Aquaculture 261: 688–694. 5. Mykles DL, Skinner DM (1990) Atrophy of crustacean somatic muscle and the proteinases that do the job. A review. J Crustacean Biol 10: 577–594. 6. Cesar JRO, Zhao B, Yang J (2008) Analysis of expressed sequenced tags from abdominal muscle cDNA library of the pacific white shrimp Litopenaeus vannamei. Animal. pp 1–7. 7. Uy R, Wold F (1977) Posttranslational covalent modification of proteins. Science 198: 890–896. A Novel Shrimp DNA-Binding Protein LvDBP23 Western blot analysis The primary antibody were custom-produced by using the synthesized peptide of amino acid position 185–198 (CVTL- DDEYTGPNDVA) of LvDBP23 protein, which was designed by online antigen software for good antigenicity and surface probability. The cytosolic and nuclear protein were subjected to electrophoresis using 15% SDS-PAGE, following by Western blotting according to the Mini Tran-Blot electrophoretic Transfer cell system (BioRad, Hercules, CA). Proteins were transferred onto PVDF membrane (BioRad, Hercules, CA) in electro blotting buffer (25 mM Tris–HCl, 190 mM Glycine, 20% methanol) at a constant current of 2.5 mA for 2 h. The membrane was immersed in blocking buffer (5% nonfat dry milk, 16 Tris buffer saline (TBS), pH 7.4, 0.1% Tween 20) at room temperature for 1 hr followed by incubation with primary antibody (rabbit polyclonal antibody, GenScript) in antibody dilution buffer (1% nonfat dry milk, 16 Tris buffer saline (TBS), pH 7.4, 0.1% Tween 20) at a concentration of 1:500 at 4 C overnight. Subsequently, the membrane was incubated in HRP-conjugated anti-rabbit IgG (Santa Cruz) for 2 h at a concentration of 1:1000, washed five time for 5 min 2 time, 10 min 2 time and 20 min 1 time with 0.1% tween-20, 16TBS and developed with Visualizer EC western blot detection kit (Millipore/Upstate, Temecula, CA). In vitro DNA binding assay The total secreted recombinant proteins (100, 200 and 400 mg) of LvDBP23-His and GFP-His were incubated with 5 ml of ssDNA-cellulose and dsDNA-cellulose beads (USB) at a concen- tration of 10 mg/ml in 20 ml of binding buffer (10 mM TRIS- HCl, pH 7.4, 2.5 mM MgCl2, 0.5% Triton X-100, and 125 mM NaCl and 10% Glycerol) with 1 ml each of 10 mg/ml Leupeptin and 1 mM Dithiothreitol (DTT). The mixture was incubated on ice at 4uC for overnight with shaking, and the beads were washed three times to remove the unbound-proteins with the 100 ml of binding buffer (without Leupeptin and DTT). After the last wash, the samples were re-suspended with 10 ml of binding buffer follow by boiling in SDS loading buffer. The amount of LvDBP23-His and GFP-His protein retained on the beads was determined by western blotting with anti-His antibody. Author Contributions Conceived and designed the experiments: YT JY AP. Performed the experiments: YT BZ JY. Analyzed the data: YT JY. Contributed reagents/ materials/analysis tools: YT JY. Wrote the paper: YT JY. 8. Meek DW, Knippschild U (2003) Posttranslational modification of MDM2. Mol Cancer Res 1: 1017–1026. 9. El Haj AJ, Houlihan DF (1987) In Vitro and In Vivo Protein Synthesis Rates in A Crustacean Muscle During the Moult Cycle. J Exp Biol 127: 413–426. 10. Cesar JRO, Yang J (2007) Expression patterns of ubiquitin, heat shock protein 70, alpha-actin and beta-actin over the molt cycle in the abdominal muscle of marine shrimp Litopenaeus vannamei. Molecular Reproduction and Develop- ment 74: 554–559. 11. Laoong-u-thai Y, Zhao B, Phongdara A, Ako H, Yang J (2009) Administration of a mutated myostatin propeptide to neonatal mice significantly enhances skeletal muscle growth. Molecular Reproduction and Development 77: 76– 82. 12. Michelson AM, Abmayr SM, Bate M, Arias AM, Maniatis T (1985) The role of calcium-dependent proteinase in molt-induced claw muscle atrophy. Prog Clin Biol Res 180: 141–150. 13. Kwak KJ, Kim YO, Kang H (2005) Characterization of transgenic Arabidopsis plants overexpressing GR-RBP4 under high salinity, dehydration, or cold stress. J Exp Biol 56: 3007–3016. PLoS ONE | www.plosone.org 7 May 2011 | Volume 6 | Issue 5 | e19959