Summary of the shrimp β-actin gene regulatory regions.

<p>The regulatory loci identified in this report are designated by ↑ when positive, and ↓ when negative. The numbers indicate the positions (the first base of starting codon ATG was set as position+1).</p

Function and Regulation Domains of a Newly Isolated Putative β-Actin Promoter from Pacific White Shrimp

<div><p>Current development of transgenic shrimp research has been hampered due to the lack of the suitable promoters and efficient transfection methods for crustaceans. A 1642 bp sequence, containing 5’-upstream sequence, exon 1, intron 1 and partial exon 2, which is responsible for transcriptional initiation of the newly reported shrimp β-actin (<i>actinT1</i>), has been isolated from the Pacific white shrimp (<i>Litopenaeus vannamei</i>) and named as SbaP. To determine its function and potential application in marine biotechnology, the sequence and functional domains were examined by constitutive expression of the luciferase reporter gene. We have identified 5’ regions that play a central role in the expression of the β-actin gene. The proximal promoter (-1642/-1325) contains two highly conserved transcriptional sites, CCAAT box and CArG motif. Two negative (-1140/-924, -222/-21) and one positive (-810/-425) regulatory elements have been identified in intron1. Transient transfection assay with a construct containing proximal promoter and enhancer (SbaPΔ-222/+1Δ-1325/-924) regions of the shrimp β-actin coupled with luciferase and <i>EGFP</i> (enhanced green fluorescent protein) showed that the promoter was not only functional in sf21 cells, but promoter activity was more than 8-fold higher than a viral-origin promoter (ie1, white spot syndrome virus immediate early gene promoter). Furthermore, SbaPΔ-222/+1Δ-1325/-924 drove a successful expression of luciferase injection assay in vivo injection and also showed higher promoter activity than the ie1 promoter, suggesting that the expression vectors constructed with SbaPΔ-222/+1Δ-1325/-924 have important potential in gene transfer studies for shrimp and other crustacean species.</p></div

Expression of Luciferase in vivo at day 2 post injection.

<p>(A) Relative transcription of luciferase in muscle after different group injection. 1: SbaP group; 2: SbaP (Δ-222/+1Δ-1325/-924) group; 3: ie1 group. (B) Real time RT-PCR products. The products were run in 2% agarose, products in upper and lower line were amplified using detection primer pairs (RT-LucF/R) and internal control primer pairs (RT-18SF/R) respectively. 1: SbaP group; 2: SbaP (Δ-222/+1Δ-1325/-924) group; 3: ie1 group; 4: negative control, pGL group.</p

Expression of EGFP driven by SbaPΔ-222/+1Δ-1325/-924 in sf21 cells.

<p>Sf21 cells were transfected with pGL-SbaPΔ-222/+1Δ-1325/-924-EGFP (a, f), pGL-SbaP-EGFP (b, g) and pGL-ie1-EGFP (c, h), pGL-Polh-EGFP (d, i) and pGL (e, j) were observed under a fluorescence microscope at day 2 post transfection. The green fluorescence protein gene (EGFP) can be detected in a, b, c (positive control), but not in d and e (negative control). The nuclei were stained with DAPI dye. Bar = 100 μm.</p

Examination of activity of 5’-upsream sequences and 1^st intron of the shrimp β-actin gene based on a reporter assay.

<p>A: schematic diagram of promoter region of luciferase reporter gene constructs. Showing various 5’-flanking sequence sequences of shrimp β-actin gene fused with luciferase gene. B: the relative levels of reporter gene expression in sf21 cells are shown. The constructs were transiently co-transfected into cells along with pRL-tk control vector. The activity of firely and Renilla luciferase in the cell lysate were measured using Dual-luciferase reporter assay (Promega) at 48h post transfection. Firefly luciferase activity was normalized to Renilla luciferase activity. The Bars indicated mean ±S.D. of luciferase activity (n = 3).</p

Characterization of regulatory region of 5’-flanking sequences of the shrimp β-actin gene based on the reporter assay.

<p>A: schematic diagram of series deletion constructs with luciferase reporter gene, which were made as described in Materials and Methods. Δ indicates a deletion; B-D: the relative levels of reporter gene expression in sf21 cells are shown. The constructs were transiently co-transfected into cells along with pRL-tk control vector. The activity of firely and Renilla luciferase in the cell lysate were measured using Dual-luciferase reporter assay (Promega) at 48h post transfection. Firefly luciferase activity was normalized to Renilla luciferase activity. The Bars indicated mean ±S.D. of luciferase activity (n = 3). *indicates statistical significance (P<0.01).</p

List of primer pairs used in construction, the restriction enzyme sites were underlined.

<p>List of primer pairs used in construction, the restriction enzyme sites were underlined.</p

List of primer pairs used in iPCR.

<p>PCR conditions: initial 3 minute 95°C denaturation, followed by 37°C of 1 min at 95°C, 30 seconds at 54°C, 4 minutes at 72°C, with a 10 minute final extension at 72°C.</p><p>List of primer pairs used in iPCR.</p

Transcriptome Analysis on Chinese Shrimp <em>Fenneropenaeus chinensis</em> during WSSV Acute Infection

<div><p>Previous studies have discovered a lot of immune-related genes responding to white spot syndrome virus (WSSV) infection in crustacean. However, little information is available in relation to underlying mechanisms of host responses during the WSSV acute infection stage in naturally infected shrimp. In this study, we employed next-generation sequencing and bioinformatic techniques to observe the transcriptome differences of the shrimp between latent infection stage and acute infection stage. A total of 64,188,426 Illumina reads, including 31,685,758 reads from the latent infection group and 32,502,668 reads from the acute infection group, were generated and assembled into 46,676 unigenes (mean length: 676 bp; range: 200–15,094 bp). Approximately 24,000 peptides were predicted and classified based on homology searches, gene ontology, clusters of orthologous groups of proteins, and biological pathway mapping. Among which, 805 differentially expressed genes were identified and categorized into 11 groups based on their possible function. Genes in the Toll and IMD pathways, the Ras-activated endocytosis process, the RNA interference pathway, anti-lipopolysaccharide factors and many other genes, were found to be activated in shrimp from latent infection stage to acute infection stage. The anti-bacterially proPO-activating cascade was firstly uncovered to be probably participated in antiviral process. These genes contain not only members playing function in host defense against WSSV, but also genes utilized by WSSV for its rapid proliferation. In addition, the transcriptome data provides detail information for identifying novel genes in absence of the genome database of shrimp.</p> </div

GO annotations of unigenes from the merged database of transcriptome from shrimp at LI and AI stages.

<p>Most non-redundant sequences can be divided into three major categories, including biological process (A), cellular component (B), and molecular function (C).</p