Table 1.
List of primer pairs used in iPCR.
Table 2.
List of primer pairs used in construction, the restriction enzyme sites were underlined.
Table 3.
List of restriction enzymes used in the serial-deletion.
Fig 1.
Nucleotide sequences of the fragment of a beta-actin gene isolated from the shrimp, Litopenaeus vannamei.
A: A schematic map of the genomic sequence of L.v. beta-actin and the 5’-flanking sequences. The numbers indicate the positions (the first base of starting codon ATG was set as position+1). B: sequence of β-actin gene upstream region, untranslated exon1, intron1 and partial exon2. Sequences in exons are shown in uppercase letters, whereas those in introns and flanking regions are shown in lowercase letters. Numbering of the nucleotide sequences is given at the left. Underlined boldface letters shows CAAT, CArG and TATA boxes in the 5’-flanking region. The useful restriction enzyme sites are underlined with their name.
Fig 2.
Examination of activity of 5’-upsream sequences and 1st intron of the shrimp β-actin gene based on a reporter assay.
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).
Fig 3.
Characterization of regulatory region of 5’-flanking sequences of the shrimp β-actin gene based on the reporter assay.
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).
Fig 4.
Expression of EGFP driven by SbaPΔ-222/+1Δ-1325/-924 in sf21 cells.
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.
Fig 5.
Expression of Luciferase in vivo at day 2 post injection.
(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.
Fig 6.
Putative-regulatory sequence of the β-actin gene homologues.
Sequence alignment of the proximal promoter region is shown. Black and open boxes mark conserved sequences and known factor binding sites, respectively; CCAAT-box and CArG motif. GenBank accession number of compared fish β-actin genes are as follows: Oreochromis niloticus (AY116536.1); Common carp (C. carpio) (M24113.1).
Fig 7.
Summary of the shrimp β-actin gene regulatory regions.
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).