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

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

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

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

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

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

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

Enterovirus detection in water vs. shellfish at 9 sampling locations.

<p>Enterovirus detection in water vs. shellfish at 9 sampling locations.</p

PCR Condition brackets included in optimization assay.

a<p>40-50°C was included if reported T_anneal in literature was <50°C.</p