A Novel Shrimp DNA-Binding Protein LvDBP23 Figure 4. Tissue specificity of LvDBP23 gene expression by RT-PCR. LvDBP23 mRNA was detected in abdominal muscle (AM) and swimming leg muscle (SM) but weakly in intestine (I), lymphoid (L) and Gill (G). EF-1 gene was used as a control gene. Abbreviations in the picture represented marker (M), heart (H), eyestalk (E), intestine (I), hepatopancreas (Hep), haemocyte (Cy), lymphoid (L), brain and nerve ganglion (BN), gill (G), pleopods (P), abdominal muscle (AM), swimming leg muscle (SM) and ovary (O). doi:10.1371/journal.pone.0019959.g004 plant can act as RNA binding proteins. In vitro nucleic acid- binding assays revealed the glycine-rich RNA-binding protein4 (GR-RBP4) binding sequence interacts with non-specifically to RNAs and DNAs [13]. Proteins that contain RNA-binding proteins (RBPs) and a glycine-rich region at the C-terminus (glycine-rich RNA-binding proteins, GR-RBPs) have been de- scribed in plants, and their involvement in plant stress response has been indicated by several expression analyses in maize [20]. In conclusion, we report a new shrimp mRNA named LvDBP23 identified from L vannamei abdominal muscle cDNA library. The 838 nucleotides of LvDBP23 cDNA contain a protein-coding sequence of 639 nucleotides and 183 nucleotides of 39 non-coding region with the stop codon (TAA) and polyadenylation signal (CATAAA). This LvDBP23 mRNA was highly expressed in abdominal muscle of the juvenile shrimp, along with other tissues such as swimming legs, intestine, lymphoid and gill. The mRNA sequence of LvDBP23 was successfully used for recombinant protein production in insect cell culture. LvDBP23 mRNA has a high level of expression during the intermot stage. Nuclei acid- binding assay showed LvDBP23 has both ssDNA and dsDNA binding properties, which support the predicted function of the DNA binding Gly-rich domain (amino acid 94–130). Therefore, we concluded that LvDBP23 is a novel putative DNA-bind protein, which may involve in regulation of muscle protein gene expression during the intermolt stage. Materials and Methods Animals and tissue sampling Cultured, specific pathogens free (SPF) nauplius, postlarvae and 3-months-old Litopenaeus vannamei from a local shrimp farm in Kahuku HI were transported alive in plastic bags with 1/3 water and 2/3 compressed air to the laboratory. Fresh tissues were collected for RNA and protein extraction following the procedure outlined by Laoong-u-thai et al. [11]. The tissue samples used for molting stage studies were the same lot as in previous reports [10,11]. cDNA identification and sequence analysis Previously, we have described the L. vannamei muscle cDNA library [6]. Positive muscle cDNA clones were identified by using phage lift hybridization method as described by [11]). To further identify regulatory genes for muscle growth. We believe that TGF- b proteins and muscle transcription factor play critical roles in control muscle formation and growth. Therefore, six degenerated oligonucleotide DNA probes designed by using the conserved sequences of TGF-b and muscle transcription factor (myostatin, myostatin-like genes, MyoD and myogenin) of vertebrate and invertebrate species, including H. sapiens, P. troglodytes, C. familiaris, M. musculus, R. norvegicus, G. gallus, D. melanogaster, A. gambiae and A. irradians. These oligonucleotide DNA probes were labeled with biotin (IDT, Coralville, IA). Hybridization was carried out as described [11]. After hybridization, the filters were washed in 26 SSC, 0.1% SDS two times for 15 min at room temperature and once for 15 min at 50uC of 0.16 SSC, 0.1%SDS. Blots were autoradiographed for empirically optimized exposure times by using CDP-StarH Chemiluminescence reagent as substrate for streptavidin alkaline phosphatase conjugates. The nucleotide and protein sequence similarities searches were conducted with BLAST algorithm at the National Center for Biotechnology Information (NCBI, http://www.ncbi .nlm.nih.gov/BLAST/). To get full-length cDNA sequences, we also did 59RACE experiment. The extension of 59 sequence was amplified by 59 Figure 5. Tissue specificity expression of LvDBP23 protein by Western blotting. Detections of LvDBP23 protein in different tissues of cytosolic (A) and nuclear (B) fractions were shown including heart (H), intestine (I), hepatopancreas (Hep), haemocyte (Cy), lymphoid (L), brain and nerve ganglion (BN), gill (G), abdominal muscle (AM) and swimming leg muscle (SM). Shrimp tissues were collected from 3-month juvenile shrimp. M represents the protein marker in kDa. LvDBP23 protein was detected by polyclonal primary antibody to LvDBP23 specific 15-amino acids peptide. doi:10.1371/journal.pone.0019959.g005 PLoS ONE | www.plosone.org 5 May 2011 | Volume 6 | Issue 5 | e19959